Methods and compositions for detecting gastrointestinal and other cancers

ABSTRACT

This application describes methods and compositions for detecting and treating vimentin-associated neoplasia. Differential methylation of the vimentin nucleotide sequences has been observed in vimentin-associated neoplasia such as neoplasia of the upper or lower gastrointestinal tract, pancreas, and/or bladder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.13/105,588, filed May 11, 2011, which is a continuation of U.S.application Ser. No. 12/322,202, filed Jan. 30, 2009, now U.S. Pat. No.7,964,353, which is a continuation of U.S. application Ser. No.10/920,119, filed on Aug. 16, 2004, now U.S. Pat. No. 7,485,420, whichclaims the benefit of priority of U.S. Provisional Application number60/495,064, filed on Aug. 14, 2003. The entire teachings of thereferenced applications are incorporated herein by reference in itsentirety.

FUNDING

Work described herein was supported by National Institutes of HealthGrant R01CA 67409, and U01CA88130 and U01CA15275. The United StatesGovernment has certain rights in the invention.

BACKGROUND

In 2001, over 1.2 million new cases of human cancer will be diagnosedand over 0.5 million people will die from cancer (American CancerSociety estimate). Despite this, more people than ever are living withand surviving cancer. In 1997, for example, approximately 8.9 millionliving Americans had a history of cancer (National Cancer Instituteestimate). People are more likely to survive cancer if the disease isdiagnosed at an early stage of development, since treatment at that timeis more likely to be successful. Early detection depends uponavailability of high-quality methods. Such methods are also useful fordetermining patient prognosis, selecting therapy, monitoring response totherapy and selecting patients for additional therapy. Consequently,there is a need for cancer diagnostic methods that are specific,accurate, minimally invasive, technically simple and inexpensive.

Gastrointestinal cancers affect millions of patients per year. Forexample, over 15,000 new cases of esophageal cancer were diagnosed in2010, and there were nearly as many deaths from this cancer alone.Similarly, about 21,000 new cases of stomach cancer were diagnosed in2010, and over 10,000 deaths resulted from stomach cancer. Theoccurrence of colorectal cancer (i.e., cancer of the colon or rectum) isis even higher. Approximately 40% of individuals with colorectal cancerdie. In 2011, it is estimated that there will be over 141,000 new casesof colorectal cancer (101,700 cases of colon and 39,510 cases of rectalcancer) and about 50,000 deaths (all statistics: American CancerSociety). As with other cancers, these rates can be decreased byimproved methods for diagnosis. Although methods for detecting each typeof cancer exist, the methods are not ideal. Generally, a combination ofendoscopy, isolation of cells (for example, via collection ofcells/tissues from a fluid sample or from a tissue sample), and/orimaging technologies are used to identify cancerous cells and tumors.There are also a variety of tests conducted for each specific cancer,but these have limitations. For example, colon cancer may be detectedwith digital rectal exams (i.e., manual probing of rectum by aphysician), which are relatively inexpensive, but are unpleasant and canbe inaccurate. Fecal occult blood testing (i.e., detection of blood instool) is nonspecific because blood in the stool has multiple causes.Colonoscopy and sigmoidoscopy (i.e., direct examination of the colonwith a flexible viewing instrument) are both uncomfortable for thepatient and expensive. Double-contrast barium enema (i.e., taking X-raysof barium-filled colon) is also an expensive procedure, usuallyperformed by a radiologist. Upper endoscopy, an examination of theesophagus, stomach and duodenum, usually performed by agastroenterologist, can detect neoplasias of these organs, but is alsoan uncomfortable and expensive procedure.

Because of the disadvantages of existing methods for detecting ortreating cancers, new methods are needed for cancer diagnosis andtherapy.

SUMMARY OF THE INVENTION

In certain aspects, the present invention is based in part onApplicants' discovery of a particular human genomic DNA region in whichthe cytosines within CpG dinucleotides are differentially methylated intissues from human cancers (e.g., upper gastrointestinal cancer, lowergastrointestinal cancer, pancreatic cancer, bladder cancer, and/orcancers associated with the digestive system or other organs) andunmethylated in normal human tissues. The region is referred tohereinafter as the “vimentin-methylation target regions” (e.g., SEQ IDNO: 45 in FIG. 45). The present methods are also based in part onApplicants' discovery that the levels of vimentin transcript in tissuesfrom human cancers are lower than the levels of vimentin transcript innormal tissues.

In one embodiment, the method comprises assaying for the presence ofdifferentially methylated vimentin nucleotide sequences (e.g., in thevimentin methylation target region) in a tissue sample or a bodily fluidsample from a subject. Tissue sample may be obtained from biopsies ofthe gastrointestinal tract, including but not limited to the esophagus,stomach, duodenum, rectum, colon, and terminal ileum. Tissue samples mayalso be obtained from biopsies of the bladder and/or pancreas. Tissuesamples may be obtained as a biopsy, or as a swab or brushing of thegastrointestinal tract (e.g., colon, stomach or esophagus), bladder,pancreas, or other organs believed to contain cancerous cells ortissues). Preferred bodily fluids include blood, serum, plasma, ablood-derived fraction, stool, colonic effluent, or urine. In oneembodiment, the method involves restriction enzyme/methylation-sensitivePCR. In another embodiment, the method comprises reacting DNA from thesample with a chemical compound that converts non-methylated cytosinebases (also called “conversion-sensitive” cytosines), but not methylatedcytosine bases, to a different nucleotide base. In a preferredembodiment, the chemical compound is sodium bisulfate, which convertsunmethylated cytosine bases to uracil. The compound-converted DNA isthen amplified using a methylation-sensitive polymerase chain reaction(MSP) employing primers that amplify the compound-converted DNA templateif cytosine bases within CpG dinucleotides of the DNA from the sampleare methylated. Production of a PCR product indicates that the subjecthas cancer or precancerous adenomas. Other methods for assaying for thepresence of methylated DNA are known in the art.

In another embodiment, the method comprises assaying for decreasedlevels of a vimentin transcript in the sample. Examples of such assaysinclude RT-PCR assays which employ primers that derived from the codingsequence of vimentin. The vimentin cDNA sequence can be found, forexample, in NCBI Accession No. NM_003380.

In another embodiment, the present invention provides a detection methodfor prognosis of a cancer (e.g., upper or lower gastrointestinal cancer)in a subject known to have or suspected of having cancer. Such methodcomprises assaying for the presence of methylated vimentin DNA (e.g., inthe vimentin methylation target region) in a tissue sample or bodilyfluid from the subject. In certain cases, it is expected that detectionof methylated vimentin DNA in a blood fraction is indicative of anadvanced state of cancer (e.g., gastrointestinal cancer such as coloncancer, esophagus cancer, gastric cancer, pancreatic cancer, or bladdercancer). In other cases, detection of methylated vimentin DNA in atissue or stool derived sample or sample from other bodily fluids may beindicative of a cancer that will respond to therapeutic agents thatdemethylate DNA or reactivate expression of the vimentin gene.

In another embodiment, the present invention provides a method formonitoring over time the status of cancer (e.g., gastrointestinal cancersuch as colon cancer, esophagus cancer, gastric cancer, pancreaticcancer, or bladder cancer) in a subject. The method comprises assayingfor the presence of methylated vimentin DNA (e.g., in the vimentinmethylation target region) in a tissue sample or bodily fluid taken fromthe subject at a first time and in a corresponding tissue sample orbodily fluid taken from the subject at a second time. Absence ofmethylated vimentin DNA from the tissue sample or bodily fluid taken atthe first time and presence of methylated vimentin DNA in the tissuesample or bodily fluid taken at the second time indicates that thecancer is progressing. Presence of methylated vimentin DNA in the tissuesample or bodily fluid taken at the first time and absence of methylatedvimentin DNA from the tissue sample or bodily fluid taken at the secondtime indicates that the cancer is regressing.

In another embodiment, the present invention provides a method forevaluating therapy in a subject having cancer or suspected of havingcancer (e.g., gastrointestinal cancer such as colon cancer, pancreaticcancer, or bladder cancer). The method comprises assaying for thepresence of methylated vimentin DNA (e.g., in the vimentin methylationtarget region) in a tissue sample or bodily fluid taken from the subjectprior to therapy and a corresponding bodily fluid taken from the subjectduring or following therapy. Loss of or a decrease in the levels ofmethylated vimentin DNA in the sample taken after or during therapy ascompared to the levels of methylated vimentin DNA in the sample takenbefore therapy is indicative of a positive effect of the therapy oncancer regression in the treated subject.

The present invention also relates to oligonucleotide primer sequencesfor use in assays (e.g., methylation-sensitive PCR assays or HpaIIassays) designed to detect the methylation status of the vimentin gene.

The present invention also provides a method of inhibiting or reducinggrowth of cancer cells (e.g., gastrointestinal cancer such as coloncancer, pancreatic cancer, or bladder cancer). The method comprisesincreasing the levels of the vimentin protein in cancer cells. In oneembodiment, the cells are contacted with the vimentin protein or abiologically active equivalent or fragment thereof under conditionspermitting uptake of the protein or fragment. In another embodiment, thecells are contacted with a nucleic acid encoding the vimentin proteinand comprising a promoter active in the cancer cell, wherein thepromoter is operably linked to the region encoding the vimentin protein,under conditions permitting the uptake of the nucleic acid by the cancercell. In another embodiment, the method comprises demethylating themethylated vimentin DNA, or otherwise reactivating the silenced vimentinpromoter.

In another embodiment, the application provides isolated or recombinantvimentin nucleotide sequences that are at least 80%, 85%, 90%, 95%, 98%,99% or identical to the nucleotide sequence of any one of SEQ ID NOs:2-7 and 45-50, and fragments of said sequences that are 10, 15, 20, 25,50, 100, or 150 base pairs in length wherein the vimentin nucleotidesequences are differentially methylated in a vimentin-associated diseasecell.

In another embodiment, the application provides a method for detectingcancer, comprising: a) obtaining a sample from a patient; and b)assaying said sample for the presence of methylation of nucleotidesequences within at least two genes selected from the group consistingof: vimentin, SLC5A8, HLTF, p16, and hMLH1; wherein methylation ofnucleotide sequences within the two genes is indicative of cancer. Insuch methods, the sample is a bodily fluid selected from the groupconsisting of blood, serum, plasma, a blood-derived fraction, stool,urine, and a colonic effluent. For example, the bodily fluid is obtainedfrom a subject suspected of having or is known to have cancer.

In certain aspects, the application provides a method for detectingneoplasia of the upper gastrointestinal tract, comprising: a) obtaininga human sample; and b) assaying said sample for the presence ofmethylation within a nucleotide sequence as set forth in SEQ ID NO: 2 orfragments thereof; wherein methylation of said nucleotide sequence isindicative of a neoplasia of the upper gastrointestinal tract. In someembodiments, the sample is a bodily fluid selected from the groupconsisting of blood, serum, plasma, a blood-derived fraction, stool,urine, and a colonic effluent. In certain embodiments, the sample isderived from a tissue. The tissue sample may be obtained from biopsiesof the gastrointestinal tract, including but not limited to theesophagus, stomach, duodenum, rectum, colon, and terminal ileum. Incertain embodiments, vimentin methylation may be detected in brushingsfrom the esophagus of a subject.

In some embodiments, the bodily fluid or tissue sample is obtained froma subject suspected of having or is known to have a neoplasia of theupper gastrointestinal tract. In exemplary embodiments, neoplasia of theupper gastrointestinal tract include gastric (also known as stomachcancer) and esophageal cancer. Gastric cancers are typically classifiedaccording to their cellular or tissue origin, which include glandularcells (adenocarcinoma), immune cells (lymphoma), hormone-producing cells(carcinoid), and nervous system tissues. Types of esophageal cancerinclude adenocarcinoma, squamous cell carcinoma, choriocarcinoma,lymphoma, sarcoma, and small cell cancer. In some embodiments, uppergastrointestinal neoplasia include Barrett's esophagus, Barrett'sesophagus with high grade dysplasia, adenocarcinoma of the esophagus,adenocarcinoma of the gastroesophageal junction, and adenocarcinoma ofthe stomach.

In certain embodiments, the foregoing assay comprises assaying for thepresence of methylation of the vimentin sequence of SEQ ID NO: 45. Inother embodiments, the assay comprises assaying for the presence ofmethylation of a vimentin sequence selected from SEQ ID NOs: 40-44. Insome embodiments, the assay is methylation-specific PCR.

In certain embodiments, the assay further comprises a) treating DNA fromthe sample with a compound that converts non-methylated cytosine basesin the DNA to a different base; b) amplifying a region of the compoundconverted vimentin nucleotide sequence with a forward primer and areverse primer; and c) analyzing the methylation patterns of saidvimentin nucleotide sequences.

In other embodiments, the assay further comprises a) treating DNA fromthe sample with a compound that converts non-methylated cytosine basesin the DNA to a different base; b) amplifying a region of the compoundconverted vimentin nucleotide sequence with a forward primer and areverse primer; and c) detecting the presence and/or amount of theamplified product.

In any of the foregoing embodiments, the forward primers are selectedfrom SEQ ID NOs: 14, 16, 19, 21, 23, 25, 27, 29, 31, 33, 37, 38, 39, andthe forward primers listed in FIG. 35. Additionally, the reverse primersare selected from SEQ ID NOs: 15, 17, 18, 20, 22, 24, 26, 28, 30, 32,34, 35, 36, and the reverse primers listed in FIG. 35.

In certain embodiments, the primer is selected from MSP29, MSP47, andMSP50.

In some embodiments, the compound used to treat DNA is a bisulfitecompound.

In some embodiments, the assay comprises using a methylation-specificrestriction enzyme. In certain embodiments, the methylation-specificrestriction enzyme is selected from HpaII, SmaI, SacII, EagI, MspI,BstUI, and BssHII. In other embodiments, the assay further comprises apair of primers selected from SEQ ID NOs: 8-13, and the primers listedin FIG. 35.

In some aspects, the application provides a method for detecting aneoplasia of the upper gastrointestinal tract in a subject, comprisingdetecting vimentin protein or nucleic acid expression in a sample fromthe subject. In some embodiments, the sample is a bodily fluid selectedfrom the group consisting of blood, serum, plasma, a blood-derivedfraction, stool, urine, and a colonic effluent. In other embodiments,the sample is derived from a tissue. The tissue sample may be obtainedfrom biopsies of the gastrointestinal tract, including but not limitedto the esophagus, stomach, duodenum, rectum, colon, and terminal ileum.In some embodiments, vimentin methylation may be detected in brushingsfrom the esophagus of a subject. In certain embodiments, the vimentinprotein is detected by immunoassays.

In some embodiments, the bodily fluid or tissue sample is obtained froma subject suspected of having or is known to have a neoplasia of theupper gastrointestinal tract.

In another aspect, the application provides a method for monitoring overtime a neoplasia of the upper gastrointestinal tract comprising: a)detecting the methylation status of a vimentin nucleotide sequence in asample from the subject for a first time; and b) detecting themethylation status of the vimentin nucleotide sequence in a sample fromthe same subject at a later time; wherein absence of methylation in thevimentin nucleotide sequence taken at a later time and the presence ofmethylation in the vimentin nucleotide sequence taken at the first timeis indicative of cancer regression; and wherein presence of methylationin the vimentin nucleotide sequence taken at a later time and theabsence of methylation in the vimentin nucleotide sequence taken at thefirst time is indicative of cancer progression.

In some embodiments, the sample is a bodily fluid selected from thegroup consisting of blood, serum, plasma, a blood-derived fraction,stool, urine, and a colonic effluent. In other embodiments, the sampleis derived from a tissue. The tissue sample may be obtained frombiopsies of the gastrointestinal tract, including but not limited to theesophagus, stomach, duodenum, rectum, colon, and terminal ileum. Incertain embodiments, vimentin methylation may be detected in brushingsfrom the esophagus of a subject.

In any of the foregoing aspects and embodiments, neoplasia of the uppergastrointestinal tract include gastric (also known as stomach cancer)and esophageal cancer. Gastric cancers are typically classifiedaccording to their cellular/tissue origin, which include glandular cells(adenocarcinoma), immune cells (lymphoma), hormone-producing cells(carcinoid), and nervous system tissues. Types of esophageal cancerinclude adenocarcinoma, squamous cell carcinoma, choriocarcinoma,lymphoma, sarcoma, and small cell cancer. In some embodiments, uppergastrointestinal neoplasia include Barrett's esophagus, Barrett'sesophagus with high grade dysplasia, adenocarcinoma of the esophagus,adenocarcinoma of the gastroesophageal junction, and adenocarcinoma ofthe stomach.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the position of CpG dinucleotides as balloons in the 5′genomic region of the vimentin gene (nucleotides 1-6200). Foursubdomains (A-D) of this region are tested for aberrant methylation incancer.

FIG. 1B shows the 5′ genomic sequence of the vimentin gene,corresponding to basepairs 56,123-62,340 of the AL133415 sequence (SEQID NO: 51).

FIG. 2 shows the RT-PCR results that vimentin is well expressed innormal colon cell lines, but is poorly expressed in colon cancer celllines. The vimentin expression is induced by the demethylating agent5-AzaCytidine in 9 of 12 colon cancer cell lines.

FIG. 3 illustrates the results from HpaII assays for vimentinmethylation in the C region by PCR amplification at 30 cycles (upperpanel) or 40 cycles (lower panel). The PCR reactions are performed afterno digestion (U), digestion with the methylation sensitive restrictionenzyme HpaII (H), or digestion with the methylation indifferent enzymeMsp 1 (M). Three Non-Cancer Normal tissues (NN1, NN2, and NN3) are allunmethylated, whereas 9 of 10 colon cancer cell lines all showmethylation.

FIG. 4 illustrates the results from HpaII assays for vimentinmethylation in the C region in 10 paired Normal/Tumor colon tissuesamples (N1-10, and T1-10), by PCR amplification at 40 cycles afterrestriction enzyme digestion by HpaII.

FIG. 5 illustrates the results from HpaII assays for vimentinmethylation in the C region in 22 paired Normal/Tumor colon tissuesamples (N11-32, and T11-32), by PCR amplification at 40 cycles afterrestriction enzyme digestion by HpaII.

FIGS. 6A-6D show a further diagrammatic depiction of the vimentin gene.The positions of primers for MS-PCR inside the B and C regions areindicated as MS-PCR pairs 1-5.

FIG. 7 shows the results from MS-PCR using primer pairs 1-5 which coverpartially the B and C regions of the vimentin genomic sequence. Primerpairs 1, 4, and 5 all detect vimentin methylation in normal colontissues (designated N) when assayed by MS-PCR at 40 cycles. In contrast,the primer pair 3 defines a differentially methylated region that ismethylated in vimentin non-expressing colon cancer cell lines, but notin normal colon tissues or in vimentin expressing cell line SW480.

FIG. 8 shows the results from MS-PCR using the primer pair MSP3. Nomethylation of vimentin is detected in any of the 14 normal colon tissuesamples from non-cancer resections (designated as NN) even when the MSP3reaction is run to 80 cycles of PCR by performing 2 sequential 40 cyclereactions.

FIG. 9 shows the comparison between the HpaII assays (upper rows) to theMS-PCR using MSP3 at 40 cycles (lower rows) for detecting vimentinmethylation in the C region in 10 paired Normal/Tumor colon tissuesamples.

FIG. 10 shows the MS-PCR using the MSP3 primer at 40 cycles fordetecting vimentin methylation in 20 paired Normal/Tumor colon tissuesamples (N1-20 and T1-20).

FIG. 11 shows the MS-PCR using the MSP3 primer at 40 cycles fordetecting vimentin methylation in 26 paired Normal/Tumor colon tissuesamples (N21-46 and T21-46).

FIG. 12 shows the MS-PCR using the MSP3 primer at 40 cycles fordetecting vimentin methylation in a set of colon cancer cell lines.

FIG. 13 shows primer sequences in HpaII assays for amplifying vimentinnucleotide sequences in A, C, and D regions. A. Forward PCR primerVM-HpaII-679U (SEQ ID NO: 8) and reverse PCR primer VM-HpaII-1266D (SEQID NO: 9) selectively amplify the methylated but not unmethylatedvimentin sequence in the A region, after digestion with HpaII.Unmethylated DNAs are cut by HpaII and so cannot be PCR amplified. B.Forward PCR primer VM-HpaII-1826U (SEQ ID NO: 10) and reverse PCR primerVM-HpaII-2195D (SEQ ID NO: 11) selectively amplify the methylated butnot unmethylated vimentin sequence in the C region, after digestion withHpaII. C. Forward PCR primer VM-HpaII-2264U (SEQ ID NO: 12) and reversePCR primer VM-HpaII-2695D (SEQ ID NO: 13) selectively amplify themethylated but not unmethylated vimentin sequence in the D region, afterdigestion with HpaII.

FIG. 14 shows the sequences of the MSP-PCR primer sets 1-5 for detectingvimentin methylation. MSP1, MSP1-2, and MSP3 are primer sets foramplifying bisulfite-converted sense sequences of the duplex methylatedvimentin DNA, including forward primer VIM1374MF (SEQ ID NO: 14) andreverse primer VIM1504MR (SEQ ID NO: 15); forward primer VIM1374MF (SEQID NO: 14) and reverse primer VIM1506MR (SEQ ID NO: 18); forward primerVIM1776MF (SEQ ID NO: 23) and reverse primer VIM1982MR (SEQ ID NO: 24).MSP2 and MSPS are primer sets for amplifying bisulfite-convertedantisense sequences of the duplex methylated vimentin DNA, including:forward primer VIM1655MF(ASS) (SEQ ID NO: 19) and reverse primerVIM1797MR(ASS) (SEQ ID NO: 20); forward primer VIM1935MF(ASS) (SEQ IDNO: 27) and reverse primer VIM2094MR(ASS) (SEQ ID NO: 28). Sequencesunderlined are the control primer sets used to amplifybisulfite-converted sequences (sense or antisense) of the duplexunmethylated vimentin DNA (designated as UF or UR), including: forwardprimer VIM1368UF (SEQ ID NO: 16) and reverse primer VIM1506UR (SEQ IDNO: 17); forward primer VIM1651UF(ASS) (SEQ ID NO: 21) and reverseprimer VIM1799UR(ASS) (SEQ ID NO: 22); forward primer VIM1771UF (SEQ IDNO: 25) and reverse primer VIM1986UR (SEQ ID NO: 26); forward primerVIM1934UF(ASS) (SEQ ID NO: 29) and reverse primer VIM2089UR(ASS) (SEQ IDNO: 30).

FIG. 15 shows the sequences of the MSP-PCR primer sets 6-10 fordetecting vimentin methylation. MSP6, MSP7, MSPS, and MSPS are primersets for amplifying bisulfite-converted sense sequences of the duplexmethylated vimentin DNA, including forward primer VIM1655MF (SEQ ID NO:31) and reverse primer VIM1792MR (SEQ ID NO: 32); forward primerVIM1655MF (SEQ ID NO: 31) and reverse primer VIM1796MR (SEQ ID NO: 35);forward primer VIM1655MF (SEQ ID NO: 31) and reverse primer VIM1804MR(SEQ ID NO: 36); forward primer VIM1843MF (SEQ ID NO: 37) and reverseprimer VIM1982MR (SEQ ID NO: 24). MSP10 are primer sets for amplifyingbisulfite-converted antisense sequences of the duplex methylatedvimentin DNA, including: forward primer VIM1929MF(ASS) (SEQ ID NO: 39)and reverse primer VIM2094MR(ASS) (SEQ ID NO: 28). Sequences underlinedare the control primer sets used to amplify bisulfite-convertedsequences (sense or antisense) of the duplex unmethylated vimentin DNA(designated as UF or UR), including: forward primer VIM1651UF (SEQ IDNO: 33) and reverse primer VIM1800UR (SEQ ID NO: 34); forward primerVIM1843UR (SEQ ID NO: 38) and reverse primer VIM1986UR (SEQ ID NO: 26);forward primer VIM1934UF(ASS) (SEQ ID NO: 29) and reverse primerVIM2089UR(ASS) (SEQ ID NO: 30).

FIGS. 16A-16D show a diagrammatic depiction of the vimentin gene. A setof 10 pairs of MS-PCR primers were designed that interrogated parts ofthe vimentin B and C regions between bp 1347 and 2094. The regionsinterrogated by these primer pairs are shown schematically.

FIG. 17 shows the MS-PCR results using the 3 pairs of primer sets MSP1,MSP1-2, and MSP3 for detecting vimentin methylation in 12 non-cancernormal samples versus 12 colon cancer cell lines.

FIG. 18 shows the MS-PCR results using the 3 pairs of primer sets MSPS,MSP6, MSP7, MSPS, MSPS, and MSP10 for detecting vimentin methylation in12 non-cancer normal samples versus 12 colon cancer cell lines.

FIG. 19 shows the MS-PCR results using the 2 pairs of primer sets MSP3and MSP1-2 for detecting vimentin methylation in microdissected aberrantcrypt foci (ACF, shown as “A”).

FIG. 20 shows the amino acid sequence (SEQ ID NO: 1) of human vimentinprotein.

FIGS. 21-26 provide the definitive sequences of the vimentin 5′ genomicregion. Each figure provides sequences corresponding to basepairs56,822-58,822 of NCBI human genomic clone AL133415 that spans the 5′region of the vimentin gene encompassing regions A-D. Each figuredesignates in bold the region from basepairs 57,427-58,326 that isdifferentially methylated in colon cancer. Moreover, in each figure,specific sequences that are interrogated by MS-PCR primers areunderlined.

FIG. 21 shows the vimentin sense strand sequence, 5′ to 3′,corresponding to basepairs 56,822-58,822 of the AL133415 sequence (SEQID NO: 2). The differentially methylated region is in bold, frombasepairs 57,427-58,326 (SEQ ID NO: 45) (also see FIG. 45).

FIG. 22 shows the bisulfite converted sequence of a methylated templatederived from the vimentin genetic sense strand shown in FIG. 21 (SEQ IDNO: 3). The sequence derived from the differentially methylated regionis in bold, from basepairs 57,427-58,326 (SEQ ID NO: 46).

FIG. 23 shows the bisulfite converted sequence of an unmethylatedtemplate derived from the vimentin genetic sense strand shown in FIG. 21(SEQ ID NO: 4). The sequence derived from the differentially methylatedregion is in bold, from basepairs 57,427-58,326 (SEQ ID NO: 47).

FIG. 24 shows the vimentin antisense strand sequence (3′-5′),corresponding to basepairs 56,822-58,822 of the AL133415 sequence (SEQID NO: 5). The differentially methylated region is in bold, frombaseparis 57,427-58,326 (SEQ ID NO: 48).

FIG. 25 shows the bisulfite converted sequence of a methylated templatederived from the vimentin genetic antisense strand (3′-5′) shown in FIG.24 (SEQ ID NO: 6). The sequence derived from the differentiallymethylated region is in bold, from basepairs 57,427-58,326 (SEQ ID NO:49).

FIG. 26 shows the bisulfite converted sequence of an unmethylatedtemplate derived from the vimentin genetic antisense strand (3′-5′)shown in FIG. 24 (SEQ ID NO: 7). The sequence derived from thedifferentially methylated region is in bold, from basepairs57,427-58,326 (SEQ ID NO: 50).

FIG. 27 shows the “A region” sequence (basepairs 56799-57385 ofAL133415, SEQ ID NO: 40) as originally defined by having convenientsites for the HpaII assays. The sequence was also referred tonucleotides 679-1266 of SEQ ID NO: 51 shown in FIGS. 1A and 1B.

FIG. 28 shows the “B region” sequence (basepairs 57436-57781 ofAL133415, SEQ ID NO: 41) as originally defined by having convenientsites for the HpaII assays. The sequence was also referred tonucleotides 1317-1661 of SEQ ID NO: 51 shown in FIGS. 1A and 1B.

FIG. 29 shows the “C region” sequence (basepairs 57946-58315 ofAL133415, SEQ ID NO: 42) as originally defined by having convenientsites for the HpaII assays. The sequence was also referred tonucleotides 1826-2195 of SEQ ID NO: 51 shown in FIGS. 1A and 1B.

FIG. 30 shows the “D region” sequence (basepairs 58384-58815 ofAL133415, SEQ ID NO: 43) as originally defined by having convenientsites for the HpaII assays. The sequence was also referred tonucleotides 2264-2695 of SEQ ID NO: 51 shown in FIGS. 1A and 1B.

FIG. 31 shows the “B′ region” sequence (basepairs 57436-57945 ofAL133415, SEQ ID NO: 44), which covers the B region as well as the gapbetween B and C regions. The sequence was also referred to nucleotides1317-1825 of SEQ ID NO: 51 shown in FIGS. 1A and 1B. This B′ region alsocontains a differentially methylated region.

FIGS. 32-34 show a diagrammatic display of the vimentin 5′ genomicregion from basepairs 56700 to 58800 of NCBI human genomic sequenceentry AL133415. Boxes show the vimentin regions A, B, C, and D. Balloonsindicate CpG dinucleotides that are targets for potential methylation.Dark balloons designate CpGs that are population polymorphisms. FIG. 32designates regions A through B, and FIGS. 33-34 designates regions Cthrough D. Bars under the figures indicate regions interrogated bydifferent methylation specific PCR reactions, as numbered by MSP1-MSP50.In these figures, the primary results of the MS-PCR reactions are shownnext to the MS-PCR primers. The leftmost set of reactions are theresults of MS-PCR in 12 non-cancer normal samples; wherein a negativeresult is the preferred outcome. The rightmost set of reactions are theresults of assay of 11 colon cancer cell lines; wherein the preferredoutcome is a positive reaction.

FIGS. 35A and 35B provides the primer sequences (MSP1-MSP50) for theMS-PCR reactions summarized in FIGS. 32-34. MF indicates forwardprimers, while MR indicates reverse primers. Primers are presumed toamplify the bisulfite converted sequences of the sense genomic strand.Primers that amplify the bisulfite converted sequence of the antisensegenomic strand are indicated by (ASS). The table also provides thegenomic location corresponding to the amplified product, relative to thebasepair numbering system of clone AL133415. The table also provides thelength of the amplified fragments. Primers shaded in dark provide thebest and preferred reaction. This figure includes SEQ ID NOs: 14, 15,18, 19, 20, 23, 24, 27, 28, 31, 32, 36, 37, 39, and 52-72.

FIG. 36 demonstrates technical sensitivity and specificity of thedifferent MS-PCR assays. At far left is shown results of MS-PCRreactions performed on non-cancer normal colon tissue for either 45 or90 cycles of PCR. 90 cycle reactions were performed by taking an aliquotfrom a 45 cycle PCR reaction, diluting it into a fresh PCR reaction, andrepeating for an additional 45 cycles. For the reactions shown, theMS-PCR reactions detect no false positives in up to 90 cycles of PCR onnormal tissue. Positive control colon cancer cell lines are shownimmediately juxtaposed at right. On the far right is shown assays of thetechnical sensitivity of different MS-PCR reaction. The middle and rightmost sets of reactions show a dilution series of MS-PCR done on DNA fromVaco5, a cell line with vimentin methylation. Positive reactions areobtained down to a level of 100 picogram of input methylated Vaco5 DNA

FIG. 37 demonstrates technical sensitivity and specificity of thedifferent MS-PCR assays for additional primer sets. Column at left showsresults of assay against a panel of 11 colon cancer cell lines at 45cycles of MS-PCR. Results at the right show a column that evaluates theMS-CPR reactions at 45 and 90 cycles against a group of non-cancernormal tissues. Next shows two columns demonstrating assay of a dilutionseries in which candidate reactions are assayed against increasingdilutions of Vaco5 DNA. The best reactions, for example VIM-MSP50M, showhigh technical sensitivity for detecting most colon cancer cell lines,show low positive rates for detecting normal colon, and show highsensitivity for detecting dilutions of Vaco5 DNA down to 50 picograms ofinput DNA. The two dilution series shown at right differ in whether theyare done by admixing previously bisulfite treated normal and Vaco5 DNA(middle column) versus (rightmost column) first admixing Vaco5 andnormal DNA; diluting the mixture; and then bisulfite treating thediluted mixture.

FIG. 38 shows primary data from assays of Normal and Tumor pairs bydifferent vimentin MS-PCR reactions.

FIG. 39 shows primary data from assays of colon normal and cancer pairs,colon adenomas, and colon cancer cell lines, by different MS-PCRreactions.

FIG. 40 shows primary data from assays of colon cancer cell lines andnon-cancer normal colon samples by different MS-PCR reactions.

FIG. 41 supplements FIG. 37, further demonstrating technical sensitivityof the different MS-PCR assays for vimentin DNA methylation. Two primersets (MSP29M and MSP50M) were tested.

FIG. 42 supplements FIG. 38, further demonstrating clinical sensitivityof the different MS-PCR assays for vimentin DNA methylation. The primarydata were obtained from assays of Normal and Tumor pairs. Three primersets (MSP29M, MSP47M, and MSP50M) were used.

FIG. 43 supplements FIGS. 39 and 40, further demonstrating clinicalsensitivity of the different MS-PCR assays for vimentin DNA methylation.The primary data were obtained from assays of colon cancer cell lines,non-cancer normal colon samples (N.C.N), colon Normal/Tumor pairs, andcolon adenomas. Three primer sets (MSP29M, MSP47M, and MSP50M) wereused.

FIGS. 44A-44D provide raw data from MS-PCR with primers MSP29, MSP47,and MSP50. The data is shown in three tables for cell lines, N/T pairs,and colon adenoma samples, respectively. Methylated samples are codedred and labeled M, while unmethylated samples are coded green andlabeled U. V-MSP29, VMSP-47, and V-MSP50 are vimentin primers. H-MSP5 isa control primer (HLTF-MSP5) for comparison.

FIG. 45 shows a 5′ genomic sequence of human vimentin gene whichcorresponds to basepairs 57,427-58,326 of GenBank Accesion No. AL133415:the sense strand (SEQ ID NO: 45).

FIG. 46 shows a 5′ genomic sequence of human vimentin gene whichcorresponds to basepairs 57,427-58,326 of GenBank Accesion No. AL133415:the sense strand (bisulfite-converted/methylated) (SEQ ID NO: 46).

FIG. 47 shows a 5′ genomic sequence of human vimentin gene whichcorresponds to basepairs 57,427-58,326 of GenBank Accesion No. AL133415:the sense strand (bisulfite-converted/unmethylated) (SEQ ID NO: 47).

FIG. 48 shows a 5′ genomic sequence of human vimentin gene whichcorresponds to basepairs 57,427-58,326 of GenBank Accesion No. AL133415:the antisense strand (SEQ ID NO: 48).

FIG. 49 shows a 5′ genomic sequence of human vimentin gene whichcorresponds to basepairs 57,427-58,326 of GenBank Accesion No. AL133415:the antisense strand (bisulfite-converted/methylated) (SEQ ID NO: 49).

FIG. 50 shows a 5′ genomic sequence of human vimentin gene whichcorresponds to basepairs 57,427-58,326 of GenBank Accesion No. AL133415:the antisense strand (bisulfite-converted/unmethylated) (SEQ ID NO: 50).

FIG. 51 shows vimentin methylation in Barrett's Esophagus and esophagealneoplasias. Shown is percent of vimentin methylation relative to totalactin DNA in each sample. Circles denote individual samples. Samples inwhich no vimentin methylation was detected are depicted as having 0.1%methylated DNA, which in most cases represented the lower limit ofdetection of the assay. In a few samples in which a lesser amount ofinput DNA was available, bars raised above the 0.1% level designate theslightly higher threshold that applied as the lower limit for detectionof positive vimentin methylation. Normal: Normal Squamous Mucosa; BE:Barrett's Esophagus; HGD: High-Grade Dysplasia; EAC:EsophagealAdenocarcinoma; SCC: Squamous Cell Cancer of the Esophagus.

FIG. 52 shows percent vimentin methylation in esophageal adenocarcinomasand matched concurrent pre-cancerous Barrett's esophagus lesions inindividual patients. BE: Barrett's Esophagus; HGD: High Grade Dysplasia;EAC: Adenocarcinoma of the Esophagus.

FIG. 53 shows percent vimentin methylation in matched longitudinalesophageal neoplasias sampled over time in individual patients.

FIG. 54 shows vimentin methylation in gastric cancer. Shown is percentof vimentin methylation relative to total actin DNA in each sample.Circles denote individual samples. Samples in which no vimentinmethylation was detected are depicted as having 0.1% methylated DNA,which in most cases represented the lower limit of detection of theassay. In a few samples in which a lesser amount of input DNA wasavailable, bars raised above the 0.1% level designate the slightlyhigher threshold that applied as the lower limit for detection ofpositive vimentin methylation.. Normal-1: Normal gastric mucosa fromcancer free cases; Normal-2: Normal gastric mucosa from cases withconcurrent gastric cancers; Signet Ring Cancer: Signet Ring GastricCancer samples; Intestinal Type Cancer: Intestinal Type Gastric Cancersamples.

FIG. 55 shows the results of vimentin DNA methylation measured inesophageal brushings taken from patients with Barrett's Esophagus (BE),Barrett's Esophagus with High Grade Dysplasia (HGD), and EsophagealAdenocarcinomas (EAC).

FIG. 56 shows the results of vimentin DNA methylation measured inpancreas cancers. The x-axis indicates case identification numbers andorigin of the samples (either pancreas cores or pancreas slides thathave been obtained from biopsied samples taken from cancer patients).The y-axis indicates the percentage of vimentin DNA methylation.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “adenoma” is used herein to describe any precancerousneoplasia or benign tumor of epithelial tissue, for example, aprecancerous neoplasia of the gastrointestinal tract, pancreas, and/orthe bladder.

The term “colon adenoma” and “polyp” are used herein to describe anyprecancerous neoplasia of the colon.

The term “blood-derived fraction” herein refers to a component orcomponents of whole blood. Whole blood comprises a liquid portion (i.e.,plasma) and a solid portion (i.e., blood cells). The liquid and solidportions of blood are each comprised of multiple components; e.g.,different proteins in plasma or different cell types in the solidportion. One of these components or a mixture of any of these componentsis a blood-derived fraction as long as such fraction is missing one ormore components found in whole blood.

The term “colon” as used herein is intended to encompass the right colon(including the cecum), the transverse colon, the left colon, and therectum.

The terms “colorectal cancer” and “colon cancer” are usedinterchangeably herein to refer to any cancerous neoplasia of the colon(including the rectum, as defined above).

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The terms “compound”, “test compound,” “agent”, and “molecule” are usedherein interchangeably and are meant to include, but are not limited to,peptides, nucleic acids, carbohydrates, small organic molecules, naturalproduct extract libraries, and any other molecules (including, but notlimited to, chemicals, metals, and organometallic compounds).

The term “compound-converted DNA” herein refers to DNA that has beentreated or reacted with a chemical compound that converts unmethylated Cbases in DNA to a different nucleotide base. For example, one suchcompound is sodium bisulfite, which converts unmethylated C to U. If DNAthat contains conversion-sensitive cytosine is treated with sodiumbisulfite, the compound-converted DNA will contain U in place of C. Ifthe DNA which is treated with sodium bisulfite contains onlymethylcytosine, the compound-converted DNA will not contain uracil inplace of the methylcytosine.

The term “de-methylating agent” as used herein refers agents thatrestore activity and/or gene expression of target genes silenced bymethylation upon treatment with the agent. Examples of such agentsinclude without limitation 5-azacytidine and 5-aza-2′-deoxycytidine.

As used herein, the phrase “gene expression” or “protein expression”includes any information pertaining to the amount of gene transcript orprotein present in a sample, as well as information about the rate atwhich genes or proteins are produced or are accumulating or beingdegraded (e.g., reporter gene data, data from nuclear runoffexperiments, pulse-chase data etc.). Certain kinds of data might beviewed as relating to both gene and protein expression. For example,protein levels in a cell are reflective of the level of protein as wellas the level of transcription, and such data is intended to be includedby the phrase “gene or protein expression information.” Such informationmay be given in the form of amounts per cell, amounts relative to acontrol gene or protein, in unitless measures, etc.; the term“information” is not to be limited to any particular means ofrepresentation and is intended to mean any representation that providesrelevant information. The term “expression levels” refers to a quantityreflected in or derivable from the gene or protein expression data,whether the data is directed to gene transcript accumulation or proteinaccumulation or protein synthesis rates, etc.

The term “detection” is used herein to refer to any process of observinga marker, or a change in a marker (such as for example the change in themethylation state of the marker), in a biological sample, whether or notthe marker or the change in the marker is actually detected. In otherwords, the act of probing a sample for a marker or a change in themarker, is a “detection” even if the marker is determined to be notpresent or below the level of sensitivity. Detection may be aquantitative, semi-quantitative or non-quantitative observation.

The term “differentially methylated vimentin nucleotide sequence” refersto a region of the vimentin nucleotide sequence that is found to bemethylated in a vimentin-associated neoplasia such as a region of thevimentin nucleotide sequence that is found to be methylated in cancertissues or cell lines, but not methylated in the normal tissues or celllines. For example, FIG. 45 provides a vimentin region that isdifferentially methylated which corresponds to basepairs 57427-58326 ofthe NCBI AL133415 sequence (SEQ ID NO: 45). This sequence is mainlywithin the B and C regions.

“Expression vector” refers to a replicable DNA construct used to expressDNA which encodes the desired protein and which includes atranscriptional unit comprising an assembly of (1) genetic element(s)having a regulatory role in gene expression, for example, promoters,operators, or enhancers, operatively linked to (2) a DNA sequenceencoding a desired protein (in this case, a vimentin protein) which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription and translation initiation and termination sequences. Thechoice of promoter and other regulatory elements generally variesaccording to the intended host cell. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of“plasmids” which refer to circular double stranded DNA loops which, intheir vector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

In the expression vectors, regulatory elements controlling transcriptionor translation can be generally derived from mammalian, microbial, viralor insect genes. The ability to replicate in a host, usually conferredby an origin of replication, and a selection gene to facilitaterecognition of transformants may additionally be incorporated. Vectorsderived from viruses, such as retroviruses, adenoviruses, and the like,may be employed.

“Gastrointestinal neoplasia” refers to neoplasia of the upper and lowergastrointestinal tract. As commonly understood in the art, the uppergastrointestinal tract includes the esophagus, stomach, and duodenum;the lower gastrointestinal tract includes the remainder of the smallintestine and all of the large intestine.

The terms “healthy”, “normal,” and “non-neoplastic” are usedinterchangeably herein to refer to a subject or particular cell ortissue that is devoid (at least to the limit of detection) of a diseasecondition, such as a neoplasia, that is associated with vimentin such asfor example neoplasia associated with silencing of vimentin geneexpression due to methylation. These terms are often used herein inreference to tissues and cells of the upper and lower gastrointestinaltract, the pancreas, and the bladder. Thus, for the purposes of thisapplication, a patient with severe heart disease but lacking a vimentinsilencing-associated disease would be termed “healthy.”

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology andidentity can each be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When an equivalentposition in the compared sequences is occupied by the same base or aminoacid, then the molecules are identical at that position; when theequivalent site occupied by the same or a similar amino acid residue(e.g., similar in steric and/or electronic nature), then the moleculescan be referred to as homologous (similar) at that position. Expressionas a percentage of homology/similarity or identity refers to a functionof the number of identical or similar amino acids at positions shared bythe compared sequences. A sequence which is “unrelated or“non-homologous” shares less than 40% identity, preferably less than 25%identity with a sequence of the present invention. In comparing twosequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid and protein sequences ofthe present invention may be used as a “query sequence” to perform asearch against public databases to, for example, identify other familymembers, related sequences or homologs. Such searches can be performedusing the NBLAST and)(BLAST programs (version 2.0) of Altschul, et al.(1990) J Mol. Biol. 215:403-10. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to nucleic acid molecules of theinvention. BLAST protein searches can be performed with the)(BLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g.,)(BLAST and BLAST)can be used. See www.ncbi.nlm.nih.gov.

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073,1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990)). The well known Smith Watermanalgorithm may also be used to determine identity.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to.”

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules in a form which does not occur innature. Moreover, an “isolated nucleic acid” is meant to include nucleicacid fragments which are not naturally occurring as fragments and wouldnot be found in the natural state.

The term “methylation-sensitive PCR” (i.e., MSP) herein refers to apolymerase chain reaction in which amplification of thecompound-converted template sequence is performed. Two sets of primersare designed for use in MSP. Each set of primers comprises a forwardprimer and a reverse primer. One set of primers, calledmethylation-specific primers (see below), will amplify thecompound-converted template sequence if C bases in CpG dinucleotideswithin the vimentin DNA are methylated. Another set of primers, calledunmethylation-specific primers (see below), will amplify thecompound-converted template sequences if C bases in CpG dinucleotideswithin the vimentin DNA are not methylated.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single-stranded (such assense or antisense) and double-stranded polynucleotides.

“Operably linked” when describing the relationship between two DNAregions simply means that they are functionally related to each other.For example, a promoter or other transcriptional regulatory sequence isoperably linked to a coding sequence if it controls the transcription ofthe coding sequence.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

The terms “proteins” and “polypeptides” are used interchangeably herein.

A “sample” includes any material that is obtained or prepared fordetection of a molecular marker or a change in a molecular marker suchas for example the methylation state, or any material that is contactedwith a detection reagent or detection device for the purpose ofdetecting a molecular marker or a change in the molecular marker.

As used herein, “obtaining a sample” includes directly retrieving asample from a subject to be assayed, or directly retrieving a samplefrom a subject to be stored and assayed at a later time. Alternatively,a sample may be obtained via a second party. That is, a sample may beobtained via, e.g., shipment, from another individual who has retrievedthe sample, or otherwise obtained the sample.

A “subject” is any organism of interest, generally a mammalian subject,such as a mouse, and preferably a human subject.

As used herein, the term “specifically hybridizes” refers to the abilityof a nucleic acid probe/primer of the invention to hybridize to at least12, 15, 20, 25, 30, 35, 40, 45, 50 or 100 consecutive nucleotides of atarget sequence, or a sequence complementary thereto, or naturallyoccurring mutants thereof, such that it has less than 15%, preferablyless than 10%, and more preferably less than 5% background hybridizationto a cellular nucleic acid (e.g., mRNA or genomic DNA) other than thetarget gene. A variety of hybridization conditions may be used to detectspecific hybridization, and the stringency is determined primarily bythe wash stage of the hybridization assay. Generally high temperaturesand low salt concentrations give high stringency, while low temperaturesand high salt concentrations give low stringency. Low stringencyhybridization is achieved by washing in, for example, about 2.0× SSC at50° C., and high stringency is achieved with about 0.2× SSC at 50° C.Further descriptions of stringency are provided below.

As applied to polypeptides, the term “substantial sequence identity”means that two peptide sequences, when optimally aligned such as by theprograms GAP or BESTFIT using default gap, share at least 90 percentsequence identity, preferably at least 95 percent sequence identity,more preferably at least 99 percent sequence identity or more.Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For example, the substitution ofamino acids having similar chemical properties such as charge orpolarity is not likely to effect the properties of a protein. Examplesinclude glutamine for asparagine or glutamic acid for aspartic acid.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., a vimentin polypeptide), which is partly or entirelyheterologous (i.e., foreign) to the transgenic animal or cell into whichit is introduced, or, is homologous to an endogenous gene of thetransgenic animal or cell into which it is introduced, but which isdesigned to be inserted, or is inserted, into the animal's genome insuch a way as to alter the genome of the cell into which it is inserted(e.g., it is inserted at a location which differs from that of thenatural gene or its insertion results in a knockout). A vimentintransgene can include one or more transcriptional regulatory sequencesand any other nucleic acid, such as introns, that may be necessary foroptimal expression of a selected nucleic acid. A vimentin transgene caninclude a vimentin nucleotide sequence (e.g., SEQ ID NO: 2) or fragmentsthereof

“Vimentin-associated proliferative disorder” refers to a disease that isassociated with either reduced expression or over-expression of thevimentin gene.

“Vimentin-associated neoplasia” refers to neoplasia associated withreduced expression or no expression of the vimentin gene. Examples ofvimentin-associated neoplasia include gastro-intestinal neoplasia suchas colon neoplasia, esophageal neoplasia, gastric neoplasia, orpancreatic neoplasia, bladder neoplasia,etc. As one of skill in the artwould recognize, the term also includes neoplasias in which vimentin isaberrantly expressed, for example, neoplasias showing increasedexpression of vimentin relative to cells from healthy control tissue,neoplasias showing a reduction of vimentin relative to controls, orneoplasias in which the methylation of vimentin is altered relative tocontrols.

“Vimentin-methylation target regions” as used herein refer to thoseregions of vimentin that are found to be differentially methylated. Forexample, FIG. 45 discloses a vimentin region wherein certain sequencesof this region are differentially methylated (e.g., SEQ ID NO: 45).

“Vimentin-nucleotide sequence” or “vimentin-nucleic acid sequence” asused herein refers to the vimentin-genomic sequences as set forth in SEQID NOs: 2-7 and fragments thereof.

“Vimentin-silencing associated diseases” as used herein includesvimentin-associated neoplasia.

II. Overview

This application is based at least in part on the recognition thatdifferential methylation of the vimentin nucleotide sequence may beindicative of neoplasia of the upper and lower gastrointestinal tract,neoplasia of the pancreas, and/or neoplasia of the bladder. Asdemonstrated herein, aberrant vimentin methylation is a highly commonepigenetic alteration in neoplasias, for example, neoplasias that arisethroughout the gut and other organs. The present findings demonstratethat vimentin methylation may be a useful biomarker of neoplasia in boththe upper and lower gastrointestinal tract, pancreas, and/or bladder.

In certain aspects, the invention relates to methods for determiningwhether a patient is likely or unlikely to suffer from a neoplasia ofthe upper and/or lower gastrointestinal tract, pancreas, bladder, orother organs. For example, in certain aspects, the invention relates tomethods for determining whether a patient is likely or unlikely to havea colon neoplasia. A colon neoplasia is any cancerous or precancerousgrowth located in, or derived from, the colon. The colon is a portion ofthe intestinal tract that is roughly three feet in length, stretchingfrom the end of the small intestine to the rectum. Viewed in crosssection, the colon consists of four distinguishable layers arranged inconcentric rings surrounding an interior space, termed the lumen,through which digested materials pass. In order, moving outward from thelumen, the layers are termed the mucosa, the submucosa, the muscularispropria and the subserosa. The mucosa includes the epithelial layer(cells adjacent to the lumen), the basement membrane, the lamina propriaand the muscularis mucosae. In general, the “wall” of the colon isintended to refer to the submucosa and the layers outside of thesubmucosa. The “lining” is the mucosa.

Precancerous colon neoplasias are referred to as adenomas or adenomatouspolyps. Adenomas are typically small mushroom-like or wart-like growthson the lining of the colon and do not invade into the wall of the colon.Adenomas may be visualized through a device such as a colonoscope orflexible sigmoidoscope. Several studies have shown that patients whoundergo screening for and removal of adenomas have a decreased rate ofmortality from colon cancer. For this and other reasons, it is generallyaccepted that adenomas are an obligate precursor for the vast majorityof colon cancers.

When a colon neoplasia invades into the basement membrane of the colon,it is considered a colon cancer, as the term “colon cancer” is usedherein. In describing colon cancers, this specification will generallyfollow the so-called “Dukes” colon cancer staging system. Thecharacteristics that describe a cancer are generally of greatersignificance than the particular term used to describe a recognizablestage. The most widely used staging systems generally use at least oneof the following characteristics for staging: the extent of tumorpenetration into the colon wall, with greater penetration generallycorrelating with a more dangerous tumor; the extent of invasion of thetumor through the colon wall and into other neighboring tissues, withgreater invasion generally correlating with a more dangerous tumor; theextent of invasion of the tumor into the regional lymph nodes, withgreater invasion generally correlating with a more dangerous tumor; andthe extent of metastatic invasion into more distant tissues, such as theliver, with greater metastatic invasion generally correlating with amore dangerous disease state.

“Dukes A” and “Dukes B” colon cancers are neoplasias that have invadedinto the wall of the colon but have not spread into other tissues. DukesA colon cancers are cancers that have not invaded beyond the submucosa.Dukes B colon cancers are subdivided into two groups: Dukes B1 and DukesB2. “Dukes Bl” colon cancers are neoplasias that have invaded up to butnot through the muscularis propria. Dukes B2 colon cancers are cancersthat have breached completely through the muscularis propria. Over afive year period, patients with Dukes A cancer who receive surgicaltreatment (i.e. removal of the affected tissue) have a greater than 90%survival rate. Over the same period, patients with Dukes B1 and Dukes B2cancer receiving surgical treatment have a survival rate of about 85%and 75%, respectively. Dukes A, B1 and B2 cancers are also referred toas Ti, T2 and T3-T4 cancers, respectively.

“Dukes C” colon cancers are cancers that have spread to the regionallymph nodes, such as the lymph nodes of the gut. Patients with Dukes Ccancer who receive surgical treatment alone have a 35% survival rateover a five year period, but this survival rate is increased to 60% inpatients that receive chemotherapy.

“Dukes D” colon cancers are cancers that have metastasized to otherorgans. The liver is the most common organ in which metastatic coloncancer is found. Patients with Dukes D colon cancer have a survival rateof less than 5% over a five year period, regardless of the treatmentregimen.

In general, neoplasia may develop through one of at least threedifferent pathways, termed chromosomal instability, microsatelliteinstability, and the CpG island methylator phenotype (CIMP). Althoughthere is some overlap, these pathways tend to present somewhat differentbiological behavior. By understanding the pathway of tumor development,the target genes involved, and the mechanisms underlying the geneticinstability, it is possible to implement strategies to detect and treatthe different types of neoplasias.

This application is based at least in part, on the recognition thatcertain target genes may be silenced or inactivated by the differentialmethylation of CpG islands in the 5′ flanking or promoter regions of thetarget gene. CpG islands are clusters of cytosine-guanosine residues ina DNA sequence, which are prominently represented in the 5-flankingregion or promoter region of about half the genes in our genome. Inparticular, this application is based at least in part on therecognition that differential methylation of the vimentin nucleotidesequence may be indicative of neoplasia. In one aspect, this applicationdiscloses that the vimentin gene can be a common target for methylationand epigenetic gene silencing in cancer cells (e.g., a neoplasia of theupper or lower gastrointestinal tract), and may function as a candidatetumor suppressor gene.

Additionally, this application is based at least in part on therecognition that differential methylation of the vimentin nucleotidesequence may be indicative of neoplasia of the upper gastrointestinaltract including, but not limited to, esophageal adenocarcinoma, as wellas other varieties of upper gastrointestinal neoplasias as describedherein. As demonstrated herein, aberrant vimentin methylation is ahighly common epigenetic alteration in neoplasias that arise throughoutthe gut. The present findings demonstrate that vimentin methylation maybe a useful biomarker of gastrointestinal neoplasia in both the upperand lower gastrointestinal tract.

Esophageal adenocarinoma (EAC) has steadily increased in incidence overrecent decades. With an 85% mortality rate this cancer is the mostrapidly increasing cause of cancer mortality from solid tumors in theAmerican population. There has thus been substantial interest indevelopment of screening approaches for early detection of EAC and itsprecursor lesions of Barrett's esophagus (BE). However, the majority ofEACs develop in patients without prior symptoms, and current approachesof endoscopic screening of individuals with persistent symptoms ofgastro-esophageal reflux disease, combined with longitudinal screeningof those found to have BE, have accordingly not had significant impacton reducing deaths from EACs. As demonstrated herein, vimentinmethylation is a highly common and early biomarker of the BE pathway.

As further demonstrated herein, detection of vimentin methylation in 82%of signet ring and 40% of intestinal type gastric cancers makes vimentinmethylation among the most common DNA alteration associated with gastriccancer. Although gastric cancers account for fewer annual deaths in theAmerican population than do esophageal cancers, gastric cancers stillremain as a significant cause of cancer mortality. Vimentin methylationprovides a useful biomarker for early detection of this disease, and mayparticularly be of utility in combination with other methylated markersthat have also been described as detecting subsets of gastriccarcinomas.

Combined with the finding that vimentin methylation is present in up to83% of colon cancers, vimentin methylation emerges as a highly commonepigenetic accompaniment of neoplasias arising throughout thegastrointestinal tract.

Vimentin is one of the cytoskeletal proteins which form the cytoplasmicintermediate filament (IF). The cytoskeleton is composed of threedifferent classes: microfilaments, microtubules, and intermediatedfilaments. Intermediate filaments are a major component of thecytoskeleton of higher eukaryotes. Vimentin is the IF proteincharacteristic of mesenchymal cells, such as fibroblasts and endothelialcells (see, e.g., Evans, 1998, BioEssays, 20:79-86). Expression ofvimentin is developmentally regulated, suggesting important functionsfor this protein besides its roles as an intracellular scaffold.Vimentin shares structural sequence similarities with the DNA bindingregion of certain transcription factors such as c-fos, fral, CREB, andc-jun, further suggesting a regulatory role for vimentin (see, e.g.,Capetanaki, et al., 1990, Oncogene, 5:645-655). Recently, it has beendemonstrated that vimentin acts as a functional perinuclear adapter forthe cytosolic phospholipase A2, thus suggesting a role for the vimentinIF in the modulation of prostaglandin biosynthesis (see, e.g., Murakamiet al., 2000, Biochim Biophys Acta, 1488:159-66). A number of proteinshave been reported as having some interaction with vimentin, forexample: 1) filament-associated proteins such as plectin and IAF-300(Svitkina, et al., 1996, J Cell Blot, 135:991-1007; Yang, et al., 1985,J Cell Blot, 100:620-631); 2) chaperon proteins such as Hsc70 andalpha-crystallin (Lee, et al., 1995, J Cell Biol, 57:150-162; Nicholl,et al., 1994, EMBO J, 13:945-953); 3) kinases such as protein kinase C(PKC), cGMP kinase, and Yes kinase (Murti, et al., 1992, Exp Cell Res,202:36-44; Owen, et al., 1996, Exp Cell Res, 225:366-373; Pryzwansky etal., 1995, Blood, 85:222-230; Ciesielski-Treska, et al., 1996, Eur JCell Biol, 68:369-376). In addition, association of vimentin with 14-3-3proteins can be induced by treatment with the phosphatase inhibitorcalyculin A (Tzivion et al., 2000, J Biol Chem, 275:29772-8). 14-3-3proteins bind to their target through a specificserine/threonine-phosphorylated motif present on the target protein.This binding is likely a crucial step in the phosphorylation-dependentregulation of various key proteins involved in signal transduction andcell cycle control. Further, it has been shown that Cdc42Hs and RaclGTPases (two Rho family members) can control vimentin IF organizationinvolving tyrosine phosphorylation events. For example, expression ofactive Cdc42Hs and Racl led to the reorganization of the IF network,showing a perinuclear collapse (Meriane et al., 2000, J Biol Chem,275:33046-52).

As noted above, early detection of gastrointestinal neoplasia (e.g.,neoplasia of the upper and lower gastrointestinal tract) coupled withappropriate intervention, is important for increasing patient survivalrates. Present systems for screening for colon neoplasia are deficientfor a variety of reasons, including a lack of specificity and/orsensitivity (e.g., Fecal Occult Blood Test, flexible sigmoidoscopy) or ahigh cost and intensive use of medical resources (e.g., colonoscopy).Alternative systems for detection of colon neoplasia would be useful ina wide range of other clinical circumstances as well. For example,patients who receive surgical and/or pharmaceutical therapy for coloncancer may experience a relapse. It would be advantageous to have analternative system for determining whether such patients have arecurrent or relapsed neoplasia of the upper and lower gastrointestinaltract. As a further example, an alternative diagnostic system wouldfacilitate monitoring an increase, decrease or persistence of neoplasiaof the upper and lower gastrointestinal tract in a patient known to havesuch a neoplasia. A patient undergoing chemotherapy may be monitored toassess the effectiveness of the therapy.

III. Vimentin Nucleic Acids, Polypeptides, and Antibodies.

The present invention is based, at least in part, on the observationthat vimentin nucleotide sequences are differentially methylated incertain vimentin-associated neoplasia, such as neoplasia of the upper orlower gastrointestinal tract, neoplasia of the pancreas, and neoplasiaof the bladder. In one aspect, the application discloses vimentinnucleotide sequences having certain regions that are differentiallymethylated in vimentin-associated neoplasia, for example, SEQ ID NOs: 2and 45 and fragments thereof. Accordingly, in one embodiment, theapplication provides isolated or recombinant nucleotide sequences thatare at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to thedifferentially methylated nucleic acid sequences, wherein detection ofmethylation in any one of said differentially methylated nucleic acidsequences would be indicative of a vimentin-associated neoplasia such asneoplasia. One of ordinary skill in the art will appreciate thatvimentin nucleic acid sequences complementary to SEQ ID NOs: 2 and 45and variants thereof are also within the scope of this invention. Suchvariant nucleotide sequences include sequences that differ by one ormore nucleotide substitutions, additions or deletions, such as allelicvariants.

In yet other embodiments, vimentin nucleotide sequences also includenucleotide sequences that will hybridize under highly stringentconditions to the nucleotide sequences designated in SEQ ID NO: 2 or 45or fragments thereof. As discussed above, one of ordinary skill in theart will understand readily that appropriate stringency conditions whichpromote DNA hybridization can be varied. One of ordinary skill in theart will understand readily that appropriate stringency conditions whichpromote DNA hybridization can be varied. For example, one could performthe hybridization at 6.0× sodium chloride/sodium citrate (SSC) at about45° C., followed by a wash of 2.0× SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0× SSC at 50° C. to a high stringency of about 0.2× SSC at 50°C. In addition, the temperature in the wash step can be increased fromlow stringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the invention providesnucleic acids which hybridize under low stringency conditions of 6× SSCat room temperature followed by a wash at 2× SSC at room temperature.

In yet another aspect, the application provides the methylated forms ofnucleotide sequence of SEQ ID NO: 2 or 45 or fragments thereof, whereinthe cytosine bases of the CpG islands present in said sequences aremethylated. In other words, the vimentin nucleotide sequences may beeither in the methylated status (e.g., as seen in vimentin-associatedneoplasias) or in the unmethylated status (e.g., as seen in normalcells). In further embodiments, the vimentin nucleotide sequences of theinvention can be isolated, recombinant, and/or fused with a heterologousnucleotide sequence, or in a DNA library.

In addition to the differentially methylated vimentin nucleotidesequences, constitutively methylated nucleotide sequences are alsopresent in the vimentin sequence (e.g., the Alu repeats and the non-Aluconstitutively methylated region in the C region). Since constitutivelymethylated vimentin nucleotide sequences are methylated in both normalcells and cancer cells, a person skilled in the art would appreciate thesignificance of detecting the differentially methylated vimentinnucleotide sequences as provided herein.

In certain embodiments, the present invention providesbisulfate-converted vimentin template DNA sequences, for example, SEQ IDNOs: 3-4, 6-7, 46-47, and 49-50, and fragments thereof. Suchbisulfate-converted vimentin template DNA can be used for detecting themethylation status, for example, by an MSP reaction or by directsequencing. These bisulfate-converted vimentin sequences are also of usefor designing primers for MS-PCR reactions that specifically detectmethylated or unmethylated vimentin templates following bisulfiteconversion. In yet other embodiments, the bisulfite-converted vimentinnucleotide sequences of the invention also include nucleotide sequencesthat will hybridize under highly stringent conditions to any nucleotidesequence selected from SEQ ID NOs: 3-4, 6-7, 46-47, and 49-50.

In further aspects, the application provides methods for producing suchbisulfite-converted nucleotide sequences, for example, the applicationprovides methods for treating a nucleotide sequence with a bisulfiteagent such that the unmethylated cytosine bases are converted to adifferent nucleotide base such as a uracil.

In yet other aspects, the application provides oligonucleotide primersfor amplifying a region within the vimentin nucleic acid sequence of anyone of SEQ ID NOs: 8-39 or any one listed in FIG. 35. In certainaspects, a pair of the oligonucleotide primers (e.g., SEQ ID NOs: 8-13)can be used in a detection assay, such as the HpaII assay. In certainaspects, primers used in an MSP reaction can specifically distinguishbetween methylated and non-methylated vimentin DNA, for example, SEQ IDNOs: 14-39 or the primers listed in FIG. 35.

The primers of the invention have sufficient length and appropriatesequence so as to provide specific initiation of amplification ofvimentin nucleic acids. Primers of the invention are designed to be“substantially” complementary to each strand of the vimentin nucleicacid sequence to be amplified. While exemplary primers are provided inSEQ ID NOs: 8-39 and in FIG. 35, it is understood that any primers thathybridizes with the bisulfite-converted vimentin sequence of SEQ ID NO:2 or 45 are included within the scope of this invention and is useful inthe method of the invention for detecting methylated nucleic acid, asdescribed. Similarly, it is understood that any primers that would serveto amplify a methylation sensitive restriction site or sites within thedifferentially methylated region of SEQ ID NO: 2 or 45 are includedwithin the scope of this invention and is useful in the method of theinvention for detecting nucleic methylated nucleic acid, as described.

The oligonucleotide primers of the invention may be prepared by usingany suitable method, such as conventional phosphotriester andphosphodiester methods or automated embodiments thereof. In one suchautomated embodiment, diethylphosphoramidites are used as startingmaterials and may be synthesized as described by Beaucage, et al.(Tetrahedron Letters, 22:1859-1862, 1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066.

The various Sequence Identification Numbers that have been used in thisapplication are summarized below in Table I.

TABLE I Sequence Identification Numbers that have been used in thisapplication. SEQ Corre- ID sponding NO Description/Name FIG. 1 aminoacid sequence of human vimentin FIG. 20 protein. 2 5′ genomic sequenceof human vimentin gene, FIG. 21 corresponding to basepairs 56,822-58,822of AL133415, sense strand. 3 5′ genomic sequence of human vimentin gene,FIG. 22 corresponding to basepairs 56,822-58,822 of AL133415, sensestrand (bisulfite- converted/methylated). 4 5′ genomic sequence of humanvimentin gene, FIG. 23 corresponding to basepairs 56,822-58,822 ofAL133415, sense strand (bisulfite- converted/unmethylated). 5 5′ genomicsequence of human vimentin gene, FIG. 24 corresponding to basepairs56,822-58,822 of AL133415, antisense strand. 6 5′ genomic sequence ofhuman vimentin gene, FIG. 25 corresponding to basepairs 56,822-58,822 ofAL133415, antisense strand (bisulfite- converted/methylated). 7 5′genomic sequence of human vimentin gene, FIG. 26 corresponding tobasepairs 56,822-58,822 of AL133415, antisense strand (bisulfite-converted/unmethylated). 8 VM-HpaII-679U FIG. 13 9 VM-HpaII-1266D FIG.13 10 VM-HpaII-1826U FIG. 13 11 VM-HpaII-2195D FIG. 13 12 VM-HpaII-2264UFIG. 13 13 VM-HpaII-2695D FIG. 13 14 VIM1374MF FIGS. 14 and 35 15VIM1504MR FIGS. 14 and 35 16 VIM1368UF FIG. 14 17 VIM1506UR FIG. 14 18VIM1506MR FIGS. 14 and 35 19 VIM1655MF(ASS) FIGS. 14 and 35 20VIM1797MR(ASS) FIGS. 14 and 35 21 VIM1651UF(ASS) FIG. 14 22VIM1799UR(ASS) FIG. 14 23 VIM1776MF FIGS. 14 and 35 24 VIM1982MR FIGS.14 and 35 25 VIM1771UF FIG. 14 26 VIM1986UR FIG. 14 27 VIM1935MF(ASS)FIGS. 14 and 35 28 VIM2094MR(ASS) FIGS. 14 and 35 29 VIM1934UF(ASS) FIG.14 30 VIM2089UR(ASS) FIG. 14 31 VIM1655MF FIGS. 15 and 35 32 VIM1792MRFIGS. 15 and 35 33 VIM1651UF FIG. 15 34 VIM1800UR FIG. 15 35 VIM1796MRFIG. 15 36 VIM1804MR FIGS. 15 and 35 37 VIM1843MF FIGS. 15 and 35 38VIM1843UR FIG. 15 39 VIM1929MF FIGS. 15 and 35 40 A region of humanvimentin gene FIG. 27 41 B region of human vimentin gene FIG. 28 42 Cregion of human vimentin gene FIG. 29 43 D region of human vimentin geneFIG. 30 44 B′ region of human vimentin gene FIG. 31 45 5′ genomicsequence of human vimentin gene, FIG. 45 corresponding to basepairs57,427-58,326 of AL133415, sense strand. 46 5′ genomic sequence of humanvimentin gene, FIG. 46 corresponding to basepairs 57,427-58,326 ofAL133415, sense strand (bisulfite- converted/methylated). 47 5′ genomicsequence of human vimentin gene, FIG. 47 corresponding to basepairs57,427-58,326 of AL133415, sense strand (bisulfite-converted/unmethylated). 48 5′ genomic sequence of human vimentin gene,FIG. 48 corresponding to basepairs 57,427-58,326 of AL133415, antisensestrand. 49 5′ genomic sequence of human vimentin gene, FIG. 49corresponding to basepairs 57,427-58,326 of AL133415, antisense strand(bisulfite- converted/methylated). 50 5′ genomic sequence of humanvimentin gene, FIG. 50 corresponding to basepairs 57,427-58,326 ofAL133415, antisense strand (bisulfite- converted/unmethylated). 51 5′genomic sequence of the vimentin gene, FIG. 1B corresponding tobasepairs 56,123-62,340 of AL133415 sequence 52-72 All MS-PCR primersets of vimentin FIG. 35

In certain other aspects, the invention relates to vimentin nucleicacids that encode the vimentin polypeptide of SEQ ID NO: 1 and variantsthereof. Variant include sequences that differ by one or more nucleotidesubstitutions, additions or deletions, such as allelic variants; andwill, therefore, include coding sequences that differ from thenucleotide sequence of the coding sequence e.g., due to the degeneracyof the genetic code. In certain embodiments, variant nucleic acids willalso include sequences that will hybridize under highly stringentconditions to a nucleotide sequence encoding SEQ ID NO: 1.

Isolated vimentin nucleic acids which differ from the nucleic acidsencoding SEQ ID NO: 1 due to degeneracy in the genetic code are alsowithin the scope of the invention. For example, a number of amino acidsare designated by more than one triplet. Codons that specify the sameamino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject proteins will exist among mammalian cells. Oneskilled in the art will appreciate that these variations in one or morenucleotides (up to about 3-5% of the nucleotides) of the nucleic acidsencoding a particular protein may exist among individuals of a givenspecies due to natural allelic variation. Any and all such nucleotidevariations and resulting amino acid polymorphisms are within the scopeof this invention.

In certain embodiments, the recombinant vimentin nucleic acid may beoperably linked to one or more regulatory nucleotide sequences in anexpression construct. Regulatory nucleotide sequences will generally beappropriate for a host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects, the invention relates to vimentin polypeptide (SEQID NO: 1) described herein, and variants polypeptides thereof. Incertain embodiments, variant polypeptides have an amino acid sequencethat is at least 75% identical to an amino acid sequence as set forth inSEQ ID NO: 1. In other embodiments, the variant polypeptide has an aminoacid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an amino acid sequence as set forth in SEQ ID NO: 1.

In certain aspects, variant vimentin polypeptides are agonists orantagonists of the vimentin polypeptide as set forth in SEQ ID NO: 1.Variants of these polypeptides may have a hyperactive or constitutiveactivity, or, alternatively, act to prevent the tumor suppressoractivity of vimentin. For example, a truncated form lacking one or moredomain may have a dominant negative effect.

In certain aspects, isolated peptidyl portions of the vimentinpolypeptide can be obtained by screening polypeptides recombinantlyproduced from the corresponding fragment of the nucleic acid encodingthe polypeptide as set forth in SEQ ID NO: 1. In addition, fragments canbe chemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments which can function as eitheragonists or antagonists of the tumor suppressor function of vimentin.

In certain aspects, variant vimentin polypeptides comprise one or morefusion domains. Well known examples of such fusion domains include, forexample, polyhistidine, Glu-Glu, glutathione S transferase (GST),thioredoxin, protein A, protein G, and an immunoglobulin heavy chainconstant region (Fc), maltose binding protein (MBP), which areparticularly useful for isolation of the fusion polypeptide by affinitychromatography. For the purpose of affinity purification, relevantmatrices for affinity chromatography, such as glutathione-, amylase-,and nickel- or cobalt- conjugated resins are used. Many of such matricesare available in “kit” form, such as the Pharmacia GST purificationsystem and the QIAexpress™ system (Qiagen) useful with (HIS₆) fusionpartners. Another fusion domain well known in the art is greenfluorescent protein (GFP). This fusion partner serves as a fluorescent“tag” which allows the fusion polypeptide of the invention to beidentified by fluorescence microscopy or by flow cytometry. The GFP tagis useful when assessing subcellular localization of the fusion vimentinpolypeptide. The GFP tag is also useful for isolating cells whichexpress the fusion vimentin polypeptide by flow cytometric methods suchas a fluorescence activated cell sorting (FACS). Fusion domains alsoinclude “epitope tags,” which are usually short peptide sequences forwhich a specific antibody is available. Well known epitope tags forwhich specific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, thefusion domains have a protease cleavage site, such as for Factor Xa orThrombin, which allow the relevant protease to partially digest thefusion vimentin polypeptide and thereby liberate the recombinantpolypeptide therefrom. The liberated polypeptide can then be isolatedfrom the fusion partner by subsequent chromatographic separation.

Another aspect of the invention pertains to an isolated antibodyspecifically immunoreactive with an epitope of a vimentin polypeptide.For example, by using immunogens derived from a vimentin polypeptide(e.g., based on its cDNA sequences), anti-protein/anti-peptide antiseraor monoclonal antibodies can be made by standard protocols (see, forexample, Antibodies: A Laboratory Manual ed. by Harlow and Lane (ColdSpring Harbor Press: 1988)). A mammal, such as a mouse, a hamster orrabbit can be immunized with an immunogenic form of the vimentinpeptide. Techniques for conferring immunogenicity on a protein orpeptide include conjugation to carriers or other techniques well knownin the art. An immunogenic portion of a polypeptide can be administeredin the presence of adjuvant. The progress of immunization can bemonitored by detection of antibody titers in plasma or serum. StandardELISA or other immunoassays can be used with the immunogen as antigen toassess the levels of antibodies.

In certain embodiment, antibodies of the invention may be useful asdiagnostic or therapeutic agents for detecting or treatingvimentin-associated diseases.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the vimentinpolypeptide. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as describedabove for whole antibodies. For example, F(ab)₂ fragments can begenerated by treating antibody with pepsin. The resulting F(ab)₂fragments can be treated to reduce disulfide bridges to produce Fabfragments. The antibody of the invention is further intended to includebispecific, single-chain, and chimeric and humanized molecules havingaffinity for the vimentin protein. In preferred embodiments, theantibody further comprises a label attached thereto and able to bedetected, (e.g., the label can be a radioisotope, fluorescent compound,enzyme or enzyme co-factor).

IV. Assays and Drug Screening Methodologies

In certain aspects, the application provides assays and methods usingthe vimentin nucleotide sequences as molecular markers that distinguishbetween healthy cells and vimentin-associated diseased cells. Forexample, in one embodiment, the application provides methods and assaysusing the vimentin nucleotide sequences as markers that distinguishbetween healthy cells and neoplasia cells. In other embodiments, theapplication provides methods and assays using the vimentin nucleotidesequences as markers that distinguish between healthy cells and cellsderived from neoplasias of the upper and lower gastrointestinal tract.In one aspect, a molecular marker of the invention is a differentiallymethylated vimentin nucleotide sequence. In another aspect, anothermarker provided herein is the vimentin gene expression product.

In certain embodiments, the invention provides assays for detectingdifferentially methylated vimentin nucleotide sequences, such as thedifferential methylation patterns seen in the B and C regions (e.g., SEQID NO: 45). Thus, a differentially methylated vimentin nucleotidesequence, in its methylated state, can be a vimentin-associatedneoplasia-specific modification that serves as a target for detectionusing various methods described herein and the methods that are wellwithin the purview of the skilled artisan in view of the teachings ofthis application.

In certain aspects, such methods for detecting methylated vimentinnucleotide sequences are based on treatment of vimentin genomic DNA witha chemical compound which converts non-methylated C, but not methylatedC (i.e., 5mC), to a different nucleotide base. One such compound issodium bisulfite, which converts C, but not 5mC, to U. Methods forbisulfite treatment of DNA are known in the art (Herman, et al., 1996,Proc Natl Acad Sci USA, 93:9821-6; Herman and Baylin, 1998, CurrentProtocols in Human Genetics, N. E. A. Dracopoli, ed., John Wiley & Sons,2:10.6.1-10.6.10; U.S. Patent No. 5,786,146). To illustrate, when a DNAmolecule that contains unmethylated C nucleotides is treated with sodiumbisulfite to become a compound-converted DNA, the sequence of that DNAis changed (C→U). Detection of the U in the converted nucleotidesequence is indicative of an unmethylated C.

The different nucleotide base (e.g., U) present in compound-convertednucleotide sequences can subsequently be detected in a variety of ways.In a preferred embodiment, the present invention provides a method ofdetecting U in compound-converted vimentin DNA sequences by using“methylation sensitive PCR” (MSP) (see, e.g., Herman, et al., 1996,Proc. Natl. Acad. Sci. USA, 93 :9821-9826; U.S. Pat. Nos. 6,265,171;6,017,704; 6,200,756). In MSP, one set of primers (i.e., comprising aforward and a reverse primer) amplifies the compound-converted templatesequence if C bases in CpG dinucleotides within the vimentin DNA aremethylated. This set of primers is called “methylation-specificprimers.” Another set of primers amplifies the compound-convertedtemplate sequence if C bases in CpG dinucleotides within the vimentin 5′flanking sequence are not methylated. This set of primers is called“unmethylation-specific primers.”

In MS-PCR, the reactions use the compound-converted DNA from a sample ina subject. In assays for vimentin methylated DNA, methylation-specificprimers are used. In the case where C within CpG dinucleotides of thetarget sequence of the DNA are methylated, the methylation-specificprimers will amplify the compound-converted template sequence in thepresence of a polymerase and an MSP product will be produced. If Cwithin CpG dinucleotides of the target sequence of the DNA is notmethylated, the methylation-specific primers will not amplify thecompound-converted template sequence in the presence of a polymerase andan MSP product will not be produced.

It is often also useful to run a control reaction for the detection ofunmethylated vimentin DNA. The reactions uses the compound-converted DNAfrom a sample in a subject and unmethylation-specific primers are used.In the case where C within CpG dinucleotides of the target sequence ofthe DNA are unmethylated, the unmethylation specific primers willamplify the compound-converted template sequence in the presence of apolymerase and an MSP product will be produced. If C within CpGdinucleotides of the target sequence of the DNA is methylated, theunmethylation-specific primers will not amplify the compound-convertedtemplate sequence in the presence of a polymerase and an MSP productwill not be produced. Note that a biologic sample will often contain amixture of both neoplastic cells that give rise to a signal withmethylation specific primers, and normal cellular elements that giverise to a signal with unmethylation-specific primers. The unmethylspecific signal is often of use as a control reaction, but does not inthis instance imply the absence of neoplasia as indicated by thepositive signal derived from reactions using the methylation specificprimers.

Primers for an MSP reaction are derived from the compound-convertedvimentin template sequence. Herein, “derived from” means that thesequences of the primers are chosen such that the primers amplify thecompound-converted template sequence in an MSP reaction. Each primercomprises a single-stranded DNA fragment which is at least 8 nucleotidesin length. Preferably, the primers are less than 50 nucleotides inlength, more preferably from 15 to 35 nucleotides in length. Because thecompound-converted vimentin template sequence can be either the Watsonstrand or the Crick strand of the double-stranded DNA that is treatedwith sodium bisulfite, the sequences of the primers is dependent uponwhether the Watson or Crick compound-converted template sequence ischosen to be amplified in the MSP. Either the Watson or Crick strand canbe chosen to be amplified.

The compound-converted vimentin template sequence, and therefore theproduct of the MSP reaction, can be between 20 to 3000 nucleotides inlength, preferably between 50 to 500 nucleotides in length, morepreferably between 80 to 150 nucleotides in length. Preferably, themethylation-specific primers result in an MSP product of a differentlength than the MSP product produced by the unmethylation-specificprimers.

A variety of methods can be used to determine if an MSP product has beenproduced in a reaction assay. One way to determine if an MSP product hasbeen produced in the reaction is to analyze a portion of the reaction byagarose gel electrophoresis. For example, a horizontal agarose gel offrom 0.6 to 2.0% agarose is made and a portion of the MSP reactionmixture is electrophoresed through the agarose gel. Afterelectrophoresis, the agarose gel is stained with ethidium bromide. MSPproducts are visible when the gel is viewed during illumination withultraviolet light. By comparison to standardized size markers, it isdetermined if the MSP product is of the correct expected size.

Other methods can be used to determine whether a product is made in anMSP reaction. One such method is called “real-time PCR.” Real-time PCRutilizes a thermal cycler (i.e., an instrument that provides thetemperature changes necessary for the PCR reaction to occur) thatincorporates a fluorimeter (i.e. an instrument that measuresfluorescence). The real-time PCR reaction mixture also contains areagent whose incorporation into a product can be quantified and whosequantification is indicative of copy number of that sequence in thetemplate. One such reagent is a fluorescent dye, called SYBR Green I(Molecular Probes, Inc.; Eugene, Oregon) that preferentially bindsdouble-stranded DNA and whose fluorescence is greatly enhanced bybinding of double-stranded DNA. When a PCR reaction is performed in thepresence of SYBR Green I, resulting DNA products bind SYBR Green I andfluorescence. The fluorescence is detected and quantified by thefluorimeter. Such technique is particularly useful for quantification ofthe amount of the product in the PCR reaction. Additionally, the productfrom the PCR reaction may be quantitated in “real-time PCR” by the useof a variety of probes that hybridize to the product including TaqManprobes and molecular beacons. Quantitation may be on an absolute basis,or may be relative to a constitutively methylated DNA standard, or maybe relative to an unmethylated DNA standard. In one instance the ratioof methylated vimentin derived product to unmethylated derived vimentinproduct may be constructed.

Methods for detecting methylation of the vimentin DNA in this inventionare not limited to MSP, and may cover any assay for detecting DNAmethylation. Another example method for detecting methylation of thevimentin DNA is by using “methylation-sensitive” restrictionendonucleases. Such methods comprise treating the genomic DNA isolatedfrom a subject with a methylation-sensitive restriction endonuclease andthen using the restriction endonuclease-treated DNA as a template in aPCR reaction. Herein, methylation-sensitive restriction endonucleasesrecognize and cleave a specific sequence within the DNA if C baseswithin the recognition sequence are not methylated. If C bases withinthe recognition sequence of the restriction endonuclease are methylated,the DNA will not be cleaved. Examples of such methylation-sensitiverestriction endonucleases include, but are not limited to HpaII, SmaI,SacII, EagI, MspI, BstUI, and BssHII. In this technique, a recognitionsequence for a methylation-sensitive restriction endonuclease is locatedwithin the template DNA, at a position between the forward and reverseprimers used for the PCR reaction. In the case that a C base within themethylation-sensitive restriction endonuclease recognition sequence isnot methylated, the endonuclease will cleave the DNA template and a PCRproduct will not be formed when the DNA is used as a template in the PCRreaction. In the case that a C base within the methylation-sensitiverestriction endonuclease recognition sequence is methylated, theendonuclease will not cleave the DNA template and a PCR product will beformed when the DNA is used as a template in the PCR reaction.Therefore, methylation of C bases can be determined by the absence orpresence of a PCR product (Kane, et al., 1997, Cancer Res, 57:808-11).No sodium bisulfite is used in this technique.

Yet another exemplary method for detecting methylation of the vimentinDNA is called the modified MSP, which method utilizes primers that aredesigned and chosen such that products of the MSP reaction aresusceptible to digestion by restriction endonucleases, depending uponwhether the compound-converted template sequence contains CpGdinucleotides or UpG dinucleotides.

Yet other methods for detecting methylation of the vimentin DNA includethe MS-SnuPE methods. This method uses compound-converted vimentin DNAas a template in a primer extension reaction wherein the primers usedproduce a product, dependent upon whether the compound-convertedtemplate contains CpG dinucleotides or UpG dinucleotides (see e.g.,Gonzalgo, et al., 1997, Nucleic Acids Res., 25:2529-31).

Another exemplary method for detecting methylation of the vimentin DNAis called COBRA (i.e., combined bisulfite restriction analysis). Thismethod has been routinely used for DNA methylation detection and is wellknown in the art (see, e.g., Xiong, et al., 1997, Nucleic Acids Res,25:2532-4).

Another exemplary method for detecting methylation of vimentin DNArequires hybridization of a compound converted DNA to arrays thatinclude probes that hybridize to sequences derived from a methylatedvimentin template.

Another exemplary method for detecting methylation of vimentin DNAincludes precipitation of methylated DNA with antibodies that bindmethylated DNA or with other proteins that bind methylated DNA, and thendetection of vimentin DNA sequences in the precipitate. The detection ofvimentin DNA could be done by PCR based methods, by hybridization toarrays, or by other methods known to those skilled in the art.

In certain embodiments, the invention provides methods that involvedirectly sequencing the product resulting from an MSP reaction todetermine if the compound-converted vimentin template sequence containsCpG dinucleotides or UpG dinucleotides. Molecular biology techniquessuch as directly sequencing a PCR product are well known in the art.

In some embodiments, methylation of DNA may be measured as a percentageof total DNA. High levels of vimentin methylation may be 10-100%methylation, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 100% methylation. Low levels of vimentin methylation may be 0%-9.99%methylation, for example, 0%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, or 9.99%. At least some normal tissues, for example, normalesophagus samples, may not have any detectable vimentin methylation.

In alternative embodiments, the skilled artisan will appreciate that thepresent invention is based in part, on the recognition that vimentin mayfunction as a tumor suppressor gene. Accordingly, in certain aspects,the invention provides assays for detecting molecular markers thatdistinguish between healthy cells and vimentin-associated diseasedcells, such as cells derived from a neoplasia of the upper or lowergastrointestinal tract. As described above, one of the molecular markersof the present application includes that methylated vimentin nucleotidesequences. Thus, in one embodiment, assaying for the methylation statusof the vimentin nucleotide sequence can be monitored for detecting avimentin-silencing associated disease.

This application further provides another molecular marker: the vimentingene expression transcript or the gene product. Thus, in anotherembodiment, expression of the vimentin nucleic acid or protein can bemonitored for detecting a vimentin-silencing associated disease such asa neoplasia of the upper or lower gastrointestinal tract.

In certain embodiments, the invention provides detection methods byassaying the above-mentioned vimentin molecular markers so as todetermine whether a patient has or does not have a disease condition.Further, such a disease condition may be characterized by decreasedexpression of vimentin nucleic acid or protein described herein. Incertain embodiments, the invention provides methods for determiningwhether a patient is or is not likely to have a vimentin-associateddisease by detecting the expression of the vimentin nucleotidesequences. In further embodiments, the invention provides methods fordetermining whether the patient is having a relapse or determiningwhether a patient's cancer is responding to treatment.

In a preferred embodiment, the application provides method for detectingneoplasia of the upper or lower gastrointestinal tract, the pancreas,and/or the bladder. In certain embodiments, the present inventionprovides methods for detecting a neoplasia that is associated withsilencing of vimentin gene. Such methods comprise assaying for thepresence of a methylated vimentin nucleotide sequence in a sampleobtained from a subject. In other aspects, the invention relates tomethods for determining whether a patient is likely or unlikely to havee.g., a neoplasia of the upper or lower gastrointestinal tract. Infurther aspects, the invention relates to methods for monitoring, e.g.,a neoplasia of the upper or lower gastrointestinal tract in a subject.

In certain embodiments, the invention provides assays for detectingvimentin protein or nucleic acid transcript described herein. In certainembodiments, a method of the invention comprises providing a biologicalsample and probing the biological sample for the vimentin expressionwhich include protein or nucleic acid transcript of the vimentin.Information regarding the vimentin expression status, and optionally thequantitative level of the vimentin expression, may then be used to drawinferences about the nature of the biological sample and, if thebiological sample was obtained from a subject, the health state of thesubject.

In certain embodiments, a method of the invention comprises detectingthe presence of vimentin protein in a sample. Optionally, the methodinvolves obtaining a quantitative measure of the vimentin protein in thesample. In view of this specification, one of skill in the art willrecognize a wide range of techniques that may be employed to detect andoptionally quantitate the presence of a protein. In preferredembodiments, vimentin protein is detected with an antibody. In manyembodiments, an antibody-based detection assay involves bringing thesample and the antibody into contact so that the antibody has anopportunity to bind to proteins having the corresponding epitope. Inmany embodiments, an antibody-based detection assay also typicallyinvolves a system for detecting the presence of antibody-epitopecomplexes, thereby achieving a detection of the presence of the proteinshaving the corresponding epitope. Antibodies may be used in a variety ofdetection techniques, including enzyme-linked immunosorbent assays(ELISAs), immunoprecipitations, Western blots. Antibody-independenttechniques for identifying a protein may also be employed. For example,mass spectroscopy, particularly coupled with liquid chromatography,permits detection and quantification of large numbers of proteins in asample. Two-dimensional gel electrophoresis may also be used to identifyproteins, and may be coupled with mass spectroscopy or other detectiontechniques, such as N-terminal protein sequencing. RNA aptamers withspecific binding for the protein of interest may also be generated andused as a detection reagent.

Samples should generally be prepared in a manner that is consistent withthe detection system to be employed. For example, a sample to be used ina protein detection system should generally be prepared in the absenceof proteases. Likewise, a sample to be used in a nucleic acid detectionsystem should generally be prepared in the absence of nucleases. In manyinstances, a sample for use in an antibody-based detection system willnot be subjected to substantial preparatory steps. For example, urinemay be used directly, as may saliva and blood, although blood will, incertain preferred embodiments, be separated into fractions such asplasma and serum.

In certain embodiments, a method of the invention comprises detectingthe presence of a vimentin-expressed nucleic acid, such as an mRNA, in asample. Optionally, the method involves obtaining a quantitative measureof the vimentin-expressed nucleic acid in the sample. In view of thisspecification, one of skill in the art will recognize a wide range oftechniques that may be employed to detect and optionally quantitate thepresence of a nucleic acid. Nucleic acid detection systems generallyinvolve preparing a purified nucleic acid fraction of a sample, andsubjecting the sample to a direct detection assay or an amplificationprocess followed by a detection assay. Amplification may be achieved,for example, by polymerase chain reaction (PCR), reverse transcriptase(RT) and coupled RT-PCR. Detection of a nucleic acid is generallyaccomplished by probing the purified nucleic acid fraction with a probethat hybridizes to the nucleic acid of interest, and in many instances,detection involves an amplification as well. Northern blots, dot blots,microarrays, quantitative PCR, and quantitative RT-PCR are all wellknown methods for detecting a nucleic acid in a sample.

In certain embodiments, the invention provides nucleic acid probes thatbind specifically to a vimentin nucleic acid. Such probes may be labeledwith, for example, a fluorescent moiety, a radionuclide, an enzyme or anaffinity tag such as a biotin moiety. For example, the TaqMan® systememploys nucleic acid probes that are labeled in such a way that thefluorescent signal is quenched when the probe is free in solution andbright when the probe is incorporated into a larger nucleic acid.

Immunoscintigraphy using monoclonal antibodies directed at the vimentinmarker may be used to detect and/or diagnose a cancer. For example,monoclonal antibodies against the vimentin marker labeled with⁹⁹Technetium, ¹¹¹Indium, ¹²⁵Iodine-may be effectively used for suchimaging. As will be evident to the skilled artisan, the amount ofradioisotope to be administered is dependent upon the radioisotope.Those having ordinary skill in the art can readily formulate the amountof the imaging agent to be administered based upon the specific activityand energy of a given radionuclide used as the active moiety. Typically0.1-100 millicuries per dose of imaging agent, preferably 1-10millicuries, most often 2-5 millicuries are administered. Thus,compositions according to the present invention useful as imaging agentscomprising a targeting moiety conjugated to a radioactive moietycomprise 0.1-100 millicuries, in some embodiments preferably 1-10millicuries, in some embodiments preferably 2-5 millicuries, in someembodiments more preferably 1-5 millicuries.

In certain embodiments, the present invention provides drug screeningassays for identifying test compounds which potentiate the tumorsuppressor function of the vimentin gene. In one aspect, the assaysdetect test compounds which potentiate the expression level of thevimentin. In another aspect, the assays detect test compounds whichinhibit the methylation of the vimentin nucleotide sequences. In certainembodiments, drug screening assays can be generated which detect testcompounds on the basis of their ability to interfere with stability orfunction of the vimentin polypeptide. Alternatively, simple bindingassays can be used to detect compounds that inhibit or potentiate theinteraction between the vimentin polypeptide and its interacting protein(e.g., plectin, IFAP-300, Hsc70, alpha-crstallin, PKC, cGMP kinase, orYes kinase) or the binding of the vimentin polypeptide to a target DNA.

A variety of assay formats may be used and, in light of the presentdisclosure, those not expressly described herein will nevertheless beconsidered to be within the purview of ordinary skill in the art. Assayformats can approximate such conditions as vimentin expression level,methylation status of vimentin sequence, tumor suppressing activity,intermediate filament formation activity, and may be generated in manydifferent forms. In many embodiments, the invention provides assaysincluding both cell-free systems and cell-based assays which utilizeintact cells.

Compounds to be tested can be produced, for example, by bacteria, yeastor other organisms (e.g., natural products), produced chemically (e.g.,small molecules, including peptidomimetics), or produced recombinantly.The efficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison. In the control assay, the formation ofcomplexes is quantitated in the absence of the test compound.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays of the present invention which are performed in cell-freesystems, such as may be developed with purified or semi-purifiedproteins or with lysates, are often preferred as “primary” screens inthat they can be generated to permit rapid development and relativelyeasy detection of an alteration in a molecular target which is mediatedby a test compound. Moreover, the effects of cellular toxicity and/orbioavailability of the test compound can be generally ignored in the invitro system, the assay instead being focused primarily on the effect ofthe drug on the molecular target as may be manifest in an alteration ofbinding affinity with other proteins or changes in enzymatic propertiesof the molecular target.

In certain embodiments, test compounds identified from these assays maybe used in a therapeutic method for treating a vimentin-associatedproliferative disease.

Still another aspect of the application provides transgenic non-humananimals which express a heterologous vimentin gene, or which have hadone or more genomic vimentin gene(s) disrupted in at least one of thetissue or cell-types of the animal. For instance, transgenic mice thatare disrupted at their vimentin gene locus can be generated.

In another aspect, the application provides an animal model for avimentin-associated proliferative disease, which has a mis-expressedvimentin allele. For example, a mouse can be bred which has a vimentinallele deleted, or in which all or part of one or more vimentin exonsare deleted. Such a mouse model can then be used to study disordersarising from mis-expression of the vimentin gene.

Accordingly, the present application discloses transgenic animals whichare comprised of cells (of that animal) containing a vimentin transgeneand which preferably (though optionally) express an exogenous vimentinprotein in one or more cells in the animal. The vimentin transgene canencode the wild-type form of the protein, or can encode homologsthereof, including both agonists and antagonists, as well as antisenseconstructs. The vimentin transgene can include a vimentin nucleotidesequence (e.g., SEQ ID NO: 2) or fragments thereof. In preferredembodiments, the expression of the transgene is restricted to specificsubsets of cells, tissues or developmental stages utilizing, forexample, cis-acting sequences that control expression in the desiredpattern.

Genetic techniques which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that catalyzesthe genetic recombination a target sequence. As used herein, the phrase“target sequence” refers to a nucleotide sequence that is geneticallyrecombined by a recombinase. The target sequence is flanked byrecombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of the vimentin polypeptides. For example, excision of atarget sequence which interferes with the expression of a recombinantvimentin gene can be designed to activate expression of that gene. Thisinterference with expression of the protein can result from a variety ofmechanisms, such as spatial separation of the vimentin gene from thepromoter element or an internal stop codon. Moreover, the transgene canbe made wherein the coding sequence of the gene is flanked recombinaserecognition sequences and is initially transfected into cells in a 3′ to5′ orientation with respect to the promoter element. In such aninstance, inversion of the target sequence will reorient the subjectgene by placing the 5′ end of the coding sequence in an orientation withrespect to the promoter element which allow for promoter driventranscriptional activation.

In an illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al., (1992) Proc. Natl. Acad. Sci. USA89:6232-6236; Orban et al., (1992) Proc. Natl. Acad. Sci. USA89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae(O'Gorman et al., (1991) Science 251:1351-1355; PCT publication WO92/15694) can be used to generate in vivo site-specific geneticrecombination systems. Cre recombinase catalyzes the site-specificrecombination of an intervening target sequence located between loxPsequences. loxP sequences are 34 base pair nucleotide repeat sequencesto which the Cre recombinase binds and are required for Cre recombinasemediated genetic recombination. The orientation of loxP sequencesdetermines whether the intervening target sequence is excised orinverted when Cre recombinase is present (Abremski et al., (1984)1 J.Biol. Chem. 259:1509-1514); catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

V. Subjects and Samples

In certain aspects, the invention relates to a subject suspected ofhaving or has a vimentin-associated disease such as a neoplasia of theupper or lower gastrointestinal tract. Alternatively, a subject may beundergoing routine screening and may not necessarily be suspected ofhaving such a vimentin-associated disease or condition. In a preferredembodiment, the subject is a human subject, and the vimentin-associateddisease is colon neoplasia. In other embodiments, the subject is ahuman, and the vimentin-associated disease is a neoplasia of the uppergastrointestinal tract.

Assaying for vimentin markers discussed above in a sample from subjectsnot known to have, e.g., a neoplasia of the upper or lowergastrointestinal tract can aid in diagnosis of such a neoplasia in thesubject. To illustrate, detecting the methylation status of the vimentinnucleotide sequence by MSP can be used by itself, or in combination withother various assays, to improve the sensitivity and/or specificity fordetecting, e.g., a neoplasia of the upper or lower gastrointestinaltract. Preferably, such detection is made at an early stage in thedevelopment of cancer, so that treatment is more likely to be effective.

In addition to diagnosis, assaying of a vimentin marker in a sample froma subject not known to have, e.g., a neoplasia of the upper or lowergastrointestinal tract, can be prognostic for the subject (i.e.,indicating the probable course of the disease). To illustrate, subjectshaving a predisposition to develop a neoplasia of the upper or lowergastrointestinal tract may possess methylated vimentin nucleotidesequences. Assaying of vimentin markers in a sample from subjects canalso be used to select a particular therapy or therapies which areparticularly effective against, e.g., a neoplasia of the upper or lowergastrointestinal tract in the subject, or to exclude therapies that arenot likely to be effective.

Assaying of vimentin markers in samples from subjects that are known tohave, or to have had, a cancer associated with silencing of the vimentingene is also useful. For example, the present methods can be used toidentify whether therapy is effective or not for certain subjects. Oneor more samples are taken from the same subject prior to and followingtherapy, and assayed for the vimentin markers. A finding that thevimentin marker is present in the sample taken prior to therapy andabsent (or at a lower level) after therapy would indicate that thetherapy is effective and need not be altered. In those cases where thevimentin marker is present in the sample taken before therapy and in thesample taken after therapy, it may be desirable to alter the therapy toincrease the likelihood that the cancer will be eradicated in thesubject. Thus, the present method may obviate the need to perform moreinvasive procedures which are used to determine a patient's response totherapy.

Cancers frequently recur following therapy in patients with advancedcancers. In this and other instances, the assays of the invention areuseful for monitoring over time the status of a cancer associated withsilencing of the vimentin gene. For subjects in which a cancer isprogressing, a vimentin marker may be absent from some or all sampleswhen the first sample is taken and then appear in one or more sampleswhen the second sample is taken. For subjects in which cancer isregressing, a vimentin marker may be present in one or a number ofsamples when the first sample is taken and then be absent in some or allof these samples when the second sample is taken.

Samples for use with the methods described herein may be essentially anybiological material of interest. For example, a sample may be a bodilyfluid sample from a subject, a tissue sample from a subject, a solid orsemi-solid sample from a subject, a primary cell culture or tissueculture of materials derived from a subject, cells from a cell line, ormedium or other extracellular material from a cell or tissue culture, ora xenograft (meaning a sample of a cancer from a first subject, e.g., ahuman, that has been cultured in a second subject, e.g., animmuno-compromised mouse). The term “sample” as used herein is intendedto encompass both a biological material obtained directly from a subject(which may be described as the primary sample) as well as anymanipulated forms or portions of a primary sample. A sample may also beobtained by contacting a biological material with an exogenous liquid,resulting in the production of a lavage liquid containing some portionof the contacted biological material. Furthermore, the term “sample” isintended to encompass the primary sample after it has been mixed withone or more additive, such as preservatives, chelators, anti-clottingfactors, etc.

In certain embodiments, a bodily fluid sample is a blood sample. In thiscase, the term “sample” is intended to encompass not only the blood asobtained directly from the patient but also fractions of the blood, suchas plasma, serum, cell fractions (e.g., platelets, erythrocytes, andlymphocytes), protein preparations, nucleic acid preparations, etc. Incertain embodiments, a bodily fluid sample is a urine sample or acolonic effluent sample. In certain embodiments, a bodily fluid sampleis a stool sample. In some embodiments, the bodily fluid may be derivedfrom the stomach, for example, gastric secretions, acid reflux, orvomit. In other embodiments, the bodily fluid may be a fluid secreted bythe pancreas or bladder. In other embodiments, the body fluid may besaliva or spit.

In certain embodiments, a tissue sample is a biopsy taken from themucosa of the gastrointestinal tract. In other embodiments, a tissuesample is the brushings from, e.g., the esophagus of a subject.

A subject is preferably a human subject, but it is expected that themolecular markers disclosed herein, and particularly their homologs fromother animals, are of similar utility in other animals. In certainembodiments, it may be possible to detect a vimentin marker directly inan organism without obtaining a separate portion of biological material.In such instances, the term “sample” is intended to encompass thatportion of biological material that is contacted with a reagent ordevice involved in the detection process.

In certain embodiments, DNA which is used as the template in an MSPreaction is obtained from a bodily fluid sample. Examples of preferredbodily fluids are blood, serum, plasma, a blood-derived fraction, stool,colonic effluent or urine. Other body fluids can also be used. Becausethey can be easily obtained from a subject and can be used to screen formultiple diseases, blood or blood-derived fractions are especiallyuseful. For example, it has been shown that DNA alterations incolorectal cancer patients can be detected in the blood of subjects(Hibi, et al., 1998, Cancer Res, 58:1405-7). Blood-derived fractions cancomprise blood, serum, plasma, or other fractions. For example, acellular fraction can be prepared as a “buffy coat” (i.e.,leukocyte-enriched blood portion) by centrifuging 5 ml of whole bloodfor 10 min at 800 times gravity at room temperature. Red blood cellssediment most rapidly and are present as the bottom-most fraction in thecentrifuge tube. The buffy coat is present as a thin creamy whitecolored layer on top of the red blood cells. The plasma portion of theblood forms a layer above the buffy coat. Fractions from blood can alsobe isolated in a variety of other ways. One method is by taking afraction or fractions from a gradient used in centrifugation to enrichfor a specific size or density of cells.

DNA is then isolated from samples from the bodily fluids. Procedures forisolation of DNA from such samples are well known to those skilled inthe art. Commonly, such DNA isolation procedures comprise lysis of anycells present in the samples using detergents, for example. After celllysis, proteins are commonly removed from the DNA using variousproteases. RNA is removed using RNase. The DNA is then commonlyextracted with phenol, precipitated in alcohol and dissolved in anaqueous solution.

VI. Therapeutic Methods for Vimentin-associated Diseases.

Yet another aspect of this application pertains to methods of treating avimentin-associated proliferative disease which arises from reducedexpression or over-expression of the vimentin gene in cells. Suchvimentin-associated proliferative diseases (for example, a neoplasia ofthe upper or lower gastrointestinal tract) can result from a widevariety of pathological cell proliferative conditions. In certainembodiments, treatment of a vimentin-associated proliferative disorderincludes modulation of the vimentin gene expression or vimentinactivity. The term “modulate” envisions the suppression of expression ofvimentin when it is over-expressed, or augmentation of vimentinexpression when it is under-expressed.

In an embodiment, the present invention provides a therapeutic method byusing a vimentin gene construct as a part of a gene therapy protocol,such as to reconstitute the function of a vimentin protein (e.g., SEQ IDNO: 1) in a cell in which the vimentin protein is mis-expressed ornon-expressed. To illustrate, cell types which exhibit pathological orabnormal growth presumably depend at least in part on a function of avimentin protein. For example, gene therapy constructs encoding thevimentin protein can be utilized in, e.g., a neoplasia of the upper orlower gastrointestinal tract that is associated with silencing of thevimentin gene.

In certain embodiments, the invention provides therapeutic methods usingagents which induce re-expression of vimentin. Loss of vimentin geneexpression in a vimentin-associated diseased cell may be due at least inpart to methylation of the vimentin nucleotide sequence, methylationsuppressive agents such as 5-deoxyazacytidine or 5-azacytidine can beintroduced into the diseased cells. Other similar agents will be knownto those of skill in the art. In a preferred embodiment, thevimentin-associated disease is, e.g., a neoplasia of the upper or lowergastrointestinal tract associated with increased methylation of vimentinnucleotide sequences.

In certain embodiments, the invention provides therapeutic methods usinga nucleic acid approach, for example, antisense nucleic acid, ribozymesor triplex agents, to block transcription or translation of a specificvimentin mRNA, either by masking that mRNA with an antisense nucleicacid or triplex agent or by cleaving it with a ribozyme. Such disordersinclude neurodegenerative diseases, for example. Antisense nucleic acidsare DNA or RNA molecules that are complementary to at least a portion ofa specific mRNA molecule (Weintraub, Scientific American, 262:40, 1990).In the cell, the antisense nucleic acids hybridize to the correspondingmRNA, forming a double-stranded molecule. The antisense nucleic acidsinterfere with the translation of the mRNA, since the cell will nottranslate an mRNA that is double-stranded. Antisense oligomers of about15 nucleotides are preferred, since they are easily synthesized and areless likely to cause problems than larger molecules when introduced intoa target vimentin over-producing cell. Use of an oligonucleotide tostall transcription is known as the triplex strategy since the oligomerwinds around double-helical DNA, forming a three-strand helix.Therefore, these triplex compounds can be designed to recognize a uniquesite on a chosen gene (Maher, et al., Antisense Res. and Dev., 1(3):227,1991; Helene, C., Anticancer Drug Design, 6(6):569, 1991). Ribozymes areRNA molecules possessing the ability to specifically cleave othersingle-stranded RNA in a manner analogous to DNA restrictionendonucleases. Through the modification of nucleotide sequences whichencode these RNAs, it is possible to engineer molecules that recognizespecific nucleotide sequences in an RNA molecule and cleave it (Cech, J.Amer. Med. Assn., 260:3030, 1988).

The present invention also provides gene therapy for the treatment ofproliferative or immunologic disorders which are mediated by vimentinprotein. Such therapy would achieve its therapeutic effect byintroduction of the vimentin antisense polynucleotide into cells havingthe proliferative disorder. Alternatively, it may be desirable tointroduce polynucleotides encoding full-length vimentin into diseasedcells.

Delivery of antisense vimentin polynucleotide or the vimentin gene canbe achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Especially preferred fortherapeutic delivery of antisense sequences is the use of targetedliposomes. Various viral vectors which can be utilized for gene therapyas taught herein include adenovirus, herpes virus, vaccinia, or,preferably, an RNA virus such as a retrovirus. Preferably, theretroviral vector is a derivative of a murine or avian retrovirus.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). Preferably, when thesubject is a human, a vector such as the gibbon ape leukemia virus(GaLV) is utilized. A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a vimentin sequence ofinterest into the viral vector, along with another gene which encodesthe ligand for a receptor on a specific target cell, for example, thevector is target-specific. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid or aprotein. Preferred targeting is accomplished by using an antibody totarget the retroviral vector. Those skilled in the art will know of, orcan readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome or attached to a viral envelope to allow target-specific deliveryof the retroviral vector containing antisense vimentin polynucleotide orthe vimentin gene.

The invention also relates to a medicament or pharmaceutical compositioncomprising a vimentin 5′ flanking polynucleotide or a vimentin 5′flanking polynucleotide operably linked to the vimentin structural gene,respectively, in a pharmaceutically acceptable excipient or mediumwherein the medicament is used for therapy of vimentin-associated cellproliferative disorders, such as, e.g., a neoplasia of the upper orlower gastrointestinal tract.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1

1. Cell culture and 5-Azacytidine treatment.

The cultures were grown and treated as described previously (Veigl, etal., 1998, Proc. Natl. Acad. Sci. USA, 95:8698-8702). The optimaltolerated doses were determined for each treated line, and two doseswere used for some lines, ranging from 1 μg/m1 to 3 μg/ml.

2. Methylation-sensitive restriction endonuclease assays (e.g., HpaIIassays).

We examined the genomic sequence upstream of and within the vimentingene (herein referred to as 5′- vimentin genomic sequence) whichcontained a CpG dense region that could potentially be methylated (FIGS.1 and 6). To test for methylation of this CpG-rich region, we firstutilized the HpaII assays. Sample DNAs were digested with themethylation-sensitive enzyme HpaII, and then amplified by a pair of PCRprimers. When the DNA is methylated, it is resistant to the HpaIIdigestion and accordingly a PCR product is produced. On the other hand,when the DNA is unmethylated, it is susceptible to the HpaII digestionand accordingly a PCR product is not produced. The positions of the CpGdinucleotides are shown as balloons in the 5′ genomic region of thevimentin gene and four subdomains A-D of this genomic region were testedfor aberrant methylation in colon cancer (FIG. 1). The positions of thePCR primers used for the HpaII assays are also shown in FIG. 1.Sequences of the PCR primers used to amplify the A, C, and D regions inthe HpaII assays are provided in FIG. 13.

3. Reduced vimentin expression in colon cancer cells.

RT-PCR results showed that the vimentin is well expressed in normalcolon, but is scantily expressed in colon cancer cell lines (FIG. 2). Toestablish that methylation was responsible for silencing vimentin geneexpression, cell lines with vimentin DNA methylation were treated with5-azacytidine (5-azaC), a demethylating agent. As shown in FIG. 2,5-azaC treatment reactivated vimentin expression in 9 of 12 colon cancercell lines (V400, V429, V503, RCA, V5, RKO, V432, V703, and V457).

4. Vimentin is frequently methylated and silenced in colon cancer celllines.

Methylation of the vimentin genomic sequence in the C region wasdetected by HpaII assays in colon cancer cell lines (FIG. 3) or colontumors (FIGS. 4-5). PCR amplification was performed at either 30 or 40cycles after no digestion (U), digestion with the methylation sensitiverestriction enzyme HpaII (H), or digestion with the methylationindifferent enzyme Mspl (M). Three Non-Cancer Normal tissues (NN) areall unmethylated, whereas 9 of 10 colon cancer cell lines all showmethylation (FIG. 3). Methylation of the vimentin genomic sequence inthe C region was also detected in paired Normal/Tumor samples by HpaIIassays. As shown in FIGS. 4 and 5, differential methylation of vimentinin the C region was detected in 16 of 31 colon tumors after PCRamplification of 40 cycles.

Overall, HpaII assays demonstrate methylation of vimentin in the Cregion, with a sensitivity for diagnosis of colon cancer of 74% and aspecificity of 93% (2 false positive normal tissues in persons withoutcolon cancer). These results establish vimentin as a gene that isdifferentially methylated in colon cancer.

In addition, similar HpaII assays results suggested that the incidenceof aberrant methylation of the vimentin nucleotide sequence in coloncancers was lesser in the A and D regions taken as total blocks, than inthe C region. However, the B region and the 3′ portion of the A region,also remain good candidate regions, that in addition to the C region,could harbor cancer specific aberrant methylation of vimentin. Resultsof HpaII assays in the A, C, D regions in colon cancer cell lines issummarized in Table II immediately below.

TABLE II Results of HpaII assays in the A, C, D regions in colon cancercell lines. Colon cancer cell line A region assay C region assay Dregion assay V364 U U U V400 faint M M faint M V429 U M NA V503 U M USW480 U U U RCA U M U V5 M M U V6 M M U RKO M M M V432 M M NA

5. Methylation-specific PCR (MS-PCR).

500 ng DNA from each sample in a volume of 50 μl were denatured by NaOH(freshly made, final concentration, 0.2 M) at 37° C. for 15 min. Next,30 μl 10 mM hydroquinone (fresh) and 520 μl 3.0 M NaHSO4 (freshlyprepared sodium bisulfite, pH 5.0) were added, and incubated at 55° C.for 16 hrs. Modified DNA was purified using Wizard DNA Clean-Up System(Promega). The reaction was desulphonated by NaOH at a finalconcentration of 0.3 M at room temperature for 15 min and neutralized byadding 10 M NH40Ac, pH 7.0, to a final concentration of 3 M. DNA wasprecipitated with 3 volumes of absolute ethanol for 30 min at −80° C.The DNA pellet was then dissolved in distilled water to giveapproximately 10 ng/μl. Sodium bisulfite treated DNA was used as thetemplate for subsequent methylation-specific PCR.

The positions of primers for MS-PCR inside the B and C regions of thevimentin genomic sequence are indicated as MS-PCR pairs 1-5 (FIG. 6).The positions of additional MS-PCR primer pair 1-2 and MSP pairs 6-10are indicated in FIG. 16. All the primer sequences were designed basedon the vimentin 5′ genomic sequence and were specific for fully modifiedDNA. The sequences of the MSP-PCR primer sets 1, 1-2, and 3-10 are shownin FIGS. 14 and 15. Sequences of control primer sets used to amplifybisulfite-converted sequences (sense or antisense) of the duplexunmethylated vimentin DNA (designated as UF or UR), are also provided inFIGS. 14 and 15. PCR was carried out and the PCR products were run on3.0% agarose gel.

6. Improved Sensitivity and Specificity of MS-PCR for Detecting VimentinMethylation.

We further used the methylation-specific PCR technique to test formethylation of the CpG-rich region of vimentin, employing PCR primersspecific for amplification of either methylated or unmethylated DNAtemplates (FIGS. 7-12). As shown in FIG. 7, MS-PCR primer pairs 1, 4,and 5 all detected methylation in normal colon tissues when assayed byPCR at 40 cycles. In contrast, MS-PCR primer pair 3 defined adifferentially methylated region that is methylated in vimentinnon-expressing colon cancer cell lines, but not in normal colonic tissueor in vimentin expressing cell line SW480. Independent MS-PCR assaysconfirmed that that the MS-PCR primer pair MS3 detected no methylationof vimentin in any of 14 normal colon resections from non-cancerresections even when the PCR reaction was run to 80 cycles by performing2 sequential 40-cycle reactions (FIG. 8).

As shown in FIG. 9, the MS-PCR assays using the primer pair MSP3 wascompared with the HpaII assays for the methylation of vimentin in the CRegion in 10 paired Normal/Tumor samples. In these 10 cases, the MS-PCRassays using the primer pair MSP3 showed substantially improvedsensitivity and specificity for detecting vimentin methylation assummarized below in Table III. Specifically, the MSP3 primer in theMS-PCR assays shows 70% sensitivity and 90% specificity (one falsepositive with an unmethylated tumor) for detecting colon cancer.

TABLE III Comparison of sensitivity and specificity between MS-PCRassays (using the MSP3 primer pair) and HpaII assays. Normal TumorMS-PCR Assays HpaII Assays unmethylated methylated 7 4 unmethylatedunmethylated 2 3 methylated methylated 0 2 methylated unmethylated 1 1

MS-PCR assays using the MSP3 primer was further extended to the analysisof 46 paired Normal/Tumor samples as shown in FIG. 10 (samples N1-20 andT1-20) and FIG. 11 (samples N21-46 and T21-46). These 46 paired sampleswere assayed by MS-PCR of 40 cycles using the MSP3 primer formethylation (M) or unmethylation (U) of the vimentin nucleotidesequence. In these 46 cases, the MS-PCR assays using the primer pairMSP3 showed 84% sensitivity and 96% specificity for detecting coloncancer as summarized below in Table IV.

TABLE IV Sensitivity and specificity of MS-PCR assays (using the MSP3primer pair) in 46 paired Normal/Tumor samples. Normal Tumor MS-PCRAssays unmethylated methylated 37 unmethylated unmethylated 6 methylatedmethylated 1 methylated unmethylated 2

The MS-PCR reaction was further used to characterize a set of coloncancer cell lines as shown in FIG. 12. In the 39 cell line samples, theMSP3 primer used in MS-PCR assays for vimentin methylation is 82%sensitive for detecting colon cancer.

The above results indicate that the vimentin genomic sequence(nucleotides 1-6200, SEQ ID NO: 2) contains a differentially methylatedregion that is methylated in colon cancer and not in normal tissue. TheHpaII assays and the MS-PCR assays using the MSP3 primer pair can beutilized for assaying differential methylation within the vimentin 5′flank and Exon 1-Intron 1 region. Detection of methylated vimentin DNAin body fluids and excreta such as blood and stool may provide a usefulearly diagnostic of colon cancer and premalignant colon adenomas.

7. Additional Results of MS-PCR Assays for Detecting VimentinMethylation.

To further investigate the extent of differential methylation in thevimentin genomic sequence, an additional set of 6 pairs of MS-PCRprimers were designed inside the B and C regions. All the MS-PCR primersequences are shown in FIGS. 14 and 15, and their positions areillustrated in FIG. 16.

These MS-PCR primers were evaluated in a set of 12 non-cancer normalsamples versus 12 colon cancer cell lines (FIGS. 17 and 18). Asindicated by the bold designations in FIG. 14, the best performing setof primers are the originally evaluated primers MSP3, and the new primerset MSP1-2. MSP-1-2 thus identifies a new differentially methylatedregion that is within the B region.

Further, aberrant methylation of vimentin nucleotide sequence appears tobe an early event in colon neoplasia. 13 colon adenoma samples wereassayed by MS-PCR reaction using the MSP3 primer for aberrantmethylation of vimentin DNA, with results that such methylation wasdetected in 7 of 13 cases. The results are summarized below in Table V.

TABLE V MS-PCR assays (using the MSP1-2 and MSP3 primer pairs) inadenoma samples. Adenoma MSP1-2 MSP3 14-16P M M 14-25P U M 23-6P M M24-23P U U 28-3P M M 453P U U 461P U U 431P M M 493P U M 418P M M 4004696P U U 400 4828P U U 400 5426P U U 5/13 7/13

Additionally, FIG. 19 shows the results of detecting aberrant vimentinmethylation in some microdissected aberrant crypt foci (i.e., ACF,abbreviated as “A” in FIG. 19) which are microscopic early colonicneoplasms. In contrast, the vimentin methylation was not detected inmicrodissected normal tissue (abbreviated as “N” in FIG. 19) from thesame individuals.

In conclusion, the present invention discloses at least three assays ofvimentin methylation: 1) MS-PCR assays using the MSP3 primer; 2) MS-PCRassays using the MSP1-2; and 3) HpaII assays. All the assays can beemployed to identify differential methylation of the vimentin genomicsequence in cancer cells but not in normal cells. Similar assays likelycan be fashioned to other CpG sequences present within the vimentingenomic sequence. Such assays, when applied to body fluids, can be usedfor early detection of cancers such as colon cancer, precancerous colonadenoma, and for detection of individuals at increased risk fordevelopment of colon cancer due to a high load of aberrant crypt foci.

Example 2

The following experiments and data further specify specific regions andtheir sequences of vimentin whose aberrant methylation is a highfrequency marker of colon cancer. These data additionally specify assaysfor these sequences.

FIGS. 32-34 are a summary that show a diagrammatic display of thevimentin 5′ genomic region from basepairs 56700 to 58800 of NCBI humangenomic sequence entry AL133415. Boxes show the vimentin regions A, B, Cand D. Previous HpaII digestion assays had demonstrated that regions Aand D were not methylated in cancer. Accordingly, regions through C wereexhaustively interrogated with methylation specific PCR assays. Balloonson the figure indicate CpG dinucleotides that are targets for potentialmethylation. Dark balloons designate CpGs that are populationpolymorphisms. FIG. 32 designates regions A through B, and FIGS. 33-34designates regions C through D. Bars under the figure indicate regionsinterrogated by different methylation specific PCR reactions, asnumbered by MSP1-MSP50. In these figures, the primary results of theMS-PCR reactions are shown next to the bar. The leftmost set ofreactions are the results of MS-PCR in 12 non-cancer normal samples;wherein a negative result is the preferred outcome. The rightmost set ofreactions are the results of assay of 11 colon cancer cell lines;wherein the preferred outcome is a positive reaction.

The MS-PCR assays in FIGS. 32-34 were categorized into five differentgroups as determined by assays of 11 colon cancer cell lines incomparison to 12 non-cancer normal-colon samples at 45 cycles of MS-PCR.The first group (including MSP1, MSP14, MSP17 on FIG. 32; MSP3, MSP20A,MSP29, MSP30, MSP31 on FIG. 33; and MSP50 on FIG. 34) shows assays thatdetected methylation in a high percentage of colon cancer cell lines,with a strong MS-PCR gel band, and detected 0% methylation in non-cancernormal samples. The best of these reactions are further designated bybeing numerically indicated in underlined numerals, and the very best ofthese are further designated by being numerically indicated in boldunderlined numerals. The second group (including MSP8, MSP22A, MSP23,MSP24, MSP32 on FIG. 33) shows assays that detected methylation in ahigh percentage of colon cancer cell lines, with a weak MS-PCR gel band,and detected 0% methylation in non-cancer normal samples. The thirdgroup (including MSP33 on FIG. 33; and MSP35, MSP36, MSP37, MSP40,MSP41, MSP47 on FIG. 34) shows assays that detected methylation in ahigh percentage of colon cancer cell lines, with a strong MS-PCR gelband, and detected 10% of samples with methylation among non-cancernormal samples. The fourth group (including MSP21 on FIG. 33; and MSP10,MSP38, MSP39, MSP43, MSP44, MSP45 on FIG. 34) shows assays that detectedmethylation in a high percentage of colon cancer cell lines, with astrong MS-PCR gel band, and detected 20% of samples with methylationamong non-cancer normal samples. The fifth group (including MSP2, MSP6,MSP7, MSPS, MSP25A, MSP26, MSP27, MSP28 on FIG. 33; and MSPS, MSP42,MSP46, MSP48, MSP49 on FIG. 34) shows assays that detected methylationin a high percentage of colon cancer cell lines, with a strong MS-PCRgel band, and detected 30% of samples with methylation among non-cancernormal samples.

FIG. 35 provides the primer sequences for the MS-PCR reactionssummarized in FIGS. 32-34. MF indicates forward primers, while MRindicates reverse primers. Primers are presumed to amplify the bisulfiteconverted sequences of the sense genomic strand. Primers that amplifythe bisulfite converted sequence of the antisense genomic strand areindicated by (ASS). The table also provides the genomic locationcorresponding to the amplified product, relative to the basepairnumbering system of clone AL133415. The table also provides the lengthof the amplified fragments. Primers shaded in dark provide the best andpreferred reaction.

FIGS. 36-37 demonstrate technical sensitivity and specificity of thedifferent MS-PCR assays. FIG. 41 supplements FIGS. 36 and 37, with twoprimer sets (MSP29M and MSP50M) further tested.

FIG. 36 at left shows technical specificity for different MS-PCRreactions. At far left is shown results of MS-PCR reactions performed onnon-cancer normal colon tissue for either 45 or 90 cycles of PCR. 90cycle reactions were performed by taking an aliquot from a 45 cycle PCRreaction, diluting it into a fresh PCR reaction, and repeating for anadditional 45 cycles. For the reactions shown, the MS-PCR reactionsdetect no false positives in up to 90 cycles of PCR on normal tissue.Positive control colon cancer cell lines are shown immediatelyjuxtaposed at right. One the far rights is shown assay of the technicalsensitivity of different MS-PCR reaction. The middle and right most setsof reactions show a dilution series of MS-PCR done on DNA from Vaco5, acell line with vimentin methylation. Positive reactions are obtaineddown to a level of 100 picogram of input methylated Vaco5 DNA.

FIG. 37 shows similar data for additional primer sets. Column at leftshows results of assay against a panel of 11 colon cancer cell lines at45 cycles of MS-PCR. Results at the right show a column that evaluatesthe MS-CPR reactions at 45 and 90 cycles against a group of non-cancernormal tissues. Next shows two columns demonstrating assay of a dilutionseries in which candidate reactions are assayed against increasingdilutions of Vaco5 DNA. The best reactions, for example VIM-MSP50M, showhigh technical sensitivity for detecting most colon cancer cell lines,show low positive rates for detecting normal colon, and show highsensitivity for detecting dilutions of Vaco5 DNA down to 50 picograms ofinput DNA. The two dilution series shown at right differ in whether theyare done by admixing previously bisulfite treated normal and Vaco5 DNA(middle column) versus (rightmost column) first admixing Vaco5 andnormal DNA; diluting the mixture; and then bisulfite treating thediluted mixture.

The different vimentin MS-PCR primers were evaluated for detection ofmethylation in 47 colon cancer cell lines. In these assays, MSP-29 ismaximally sensitive, detecting methylation in 80% of cell lines.Increased sensitivity would be achieved by combining MSP-29 with MSP-14or MSP-17. In a separate experiment, the different vimentin MS-PCRprimers were analogously evaluated in a panel of matched colon cancertissue and paired normal colon tissue from an extensive group of coloncancer patients. Sensitivity for detection of colon cancer exceeds 85%in these assays. MSP-29 shows sensitivity of 85% with only one normalsample detected as methylated, and so is a preferred reaction. Inanother separate experiment, the different vimentin MS-PCR primers wereanalogously evaluated in a panel of 13 colon adenoma samples.Sensitivities of 62-69% are achieved for detection of aberrantmethylation in adenoma samples.

FIGS. 21-26 provide the definitive sequences of the vimentin genomicregion. Sequences are provided for the native sense and antisensevimentin genomic region, for the bisulfite converted sequences oftemplates derived from methylated and unmethylated forms of the vimentinsense strand, and for the bisulfite converted sequences of the templatesderived from the methylated and unmethylated forms of the vimentinantisense strand. Each figure provides sequences corresponding tobasepairs 56,822-58,822 of NCBI human genomic clone AL133415 that spansthe 5′ region of the vimentin gene encompassing regions A-D. Each figuredesignates in bold the region from basepairs 57,427-58,326 that we haveshown is differentially methylated in colon cancer (that is methylatedat high frequency in colon cancer and not methylated in normal colontissue). This region encompasses all of the high quality MS-PCRreactions that we have defined. Moreover, each figure underlinesspecific sequences that are interrogated by MS-PCR primers correspondingto the best MS-PCR reactions.

Specifically, FIG. 21 shows the vimentin sense strand sequence, 5′ to3′, corresponding to AL133415 sequences 56,822-58,822, with thedifferentially methylated region from 57,427-58,326 in bold. FIG. 22shows the bisulfite converted sequence of a methylated template derivedfrom the vimentin genetic sense strand corresponding to FIG. 21, withthe sequence derived from the differentially methylated region57,427-58,326 in bold. FIG. 23 shows the bisulfite converted sequence ofan unmethylated template derived from the vimentin genetic sense strandcorresponding to FIG. 21, with the sequence derived from thedifferentially methylated region 57,427-58,326 in bold. FIG. 24 showsthe vimentin antisense strand sequence, corresponding to AL133415sequences 56,822-58,822, with the differentially methylated region from57,427-58,326 in bold. Note sequence is written out 3′ to 5′. FIG. 25shows the bisulfite converted sequence of a methylated template derivedfrom the vimentin genetic antisense strand corresponding to FIG. 24,with the sequence derived from the differentially methylated region57,427-58,326 in bold. Note sequence is written out 3′ to 5′. FIG. 26shows the bisulfite converted sequence of an unmethylated templatederived from the vimentin genetic antisense strand corresponding to FIG.24, with the sequence derived from the differentially methylated region57,427-58,326 in bold. Note sequence is written out 3′ to 5′.

The above data provides the core information for the final disclosure ofthe invention of finding a region of the vimentin gene whosedifferential methylation is a specific marker for human colon cancer andprecancerous adenomas. This application also provides some additionalsupporting data as follows.

FIG. 38 shows primary data from assays of Normal and Tumor pairs bydifferent vimentin MS-PCR reactions. FIG. 42 supplements FIG. 38,further demonstrating clinical sensitivity of the MS-PCR assays usingthree primer sets (MSP29M, MSP47M, and MSP50M).

FIGS. 39 and 40 show primary data from assays on colon Normal/Tumorpairs, colon adenomas, colon cancer cell lines, and non-cancer normalcolon samples (N.C.N) by different MS-PCR reactions. FIG. 43 supplementsFIGS. 39 and 40, further demonstrating clinical sensitivity of thedifferent MS-PCR assays using three primer sets (MSP29M, MSP47M, andMSP50M).

FIG. 44 provides raw data from MS-PCR assays with three primer sets(MSP29, MSP47, and MSP50). The data are shown in three tables for celllines, N/T pairs, and colon adenoma samples, respectively. Methylatedsamples are coded red and labeled M, while unmethylated samples arecoded green and labeled U. V-MSP29, VMSP-47, and V-MSP50 are vimentinprimers. H-MSP5 is a control primer (HLTF-MSP5) for comparison. Asummary of the above sensitivity data is listed in Table VI below. Forexample, MSP29 shows 80% sensitivity for identifying cell lines (41lines tested), and 85% sensitivity for identifying tumors (46 tumorstested). MSP50 shows 73% sensitivity for identifying colon cancer celllines, and 87% sensitivity for identifying colon cancer tumors.

TABLE VI Data summary on sensitivity tests of MS-PCR based biomarkers.Cell lines Normal/Tumor pairs MS-PCR primers (source: Markowitz lab)(source: Markowitz lab) V-MSP29 33/41 (80%) 39/46 (85%) V-MSP47 30/41(73%) 40/46 (87%) V-MSP50 30/41 (73%) 40/46 (87%) H-MSP5 13/36 (36%)18/46 (39%)

In summary, the data provides a description of colon cancer and adenomaspecific aberrant methylation of vimentin gene sequences basepairs57,427-58,326 in NCBI clone AL133415, and provides MS-PCR reactions thatcan detect this aberrant methylation in a cancer specific reaction withsensitivities of about 85% as a single reaction and with sensitivitiesof about 90% in combination panels with other MS-PCR reactions.

Example 3

The following studies further examined the development of vimentinmethylation in pathologies of the upper gastrointestinal tract usingquantitative real-time-based MS-PCR for methylation detection. For thesestudies, vimentin methylation was detected in formalin fixed andparaffin embedded (FFPE) archived tissues. The assay was applied tosamples of Barrett's esophagus, esophageal adenocarinomas, and gastriccancers.

1. Tissues and FFPE DNA Preparation

Normal and neoplastic gastrointestinal tissue samples were retrieved asFFPE samples that were obtained under an IRB approved protocol. Sampleswere retrieved either as sections cut from tissue blocks or as coresprepared from tissue blocks. DNA was purified using QIAamp DNA micro kitaccording to the manufacturer's protocol with the followingmodifications: initial incubation in buffer ATL with Proteinase K wascarried out at 60° C. instead of 56° C. and proceeded for 4 days insteadof 16 hours. An additional 1.5 ml of Proteinase K was added after 3-24hours of incubation. The DNA was eluted from columns in 50 ml ofdistilled water and used immediately for bisulfite conversion, or frozenat -80° C. until use.

2. Bisulfite Conversion of the Genomic DNA and Real-time MS-PCR Assay

To create a template for methylation-specific PCR, DNA samples weresubjected to bisulfite conversion and purified using an Epitect kitaccording to the manufacturer's protocol. 4 ml of bisulfite-convertedDNA at a concentration of 0.2-25 ng/ml was used as a template forreal-time MS-PCR assay. To normalize input DNA amounts, a companionreal-time PCR assay was designed against bisulfite converted Actin genesequences that lack CpG dinucleotides and so are not modfied bymethylation. The assay for Actin was designed to generate anamplification product of the same size as the assay for methylatedvimentin. For both Actin and vimentin real-time PCR, a mixture of DNAfrom 4 colon cancer cell lines that are each fully methylated across thevimentin CpG island, was used to generate a dilution standard curve thatwas run with all real-time assays and used as part of data analysis inBioRad CFX manager software to convert the Ct values into ng DNAamounts. Vimentin methylation was calculated as percentage ratio of theamount of methylated DNA measured by vimentin PCR, divided by totalbisulfite-converted DNA amount in the sanple, as measured by Actin qPCR.The real-time MS-PCR reactions were performed in triplicates in 20 ulvolume using a LightCycler PCR master mix (Roche) with 400 nM eachprimer and 200 nM probe (sequences below). BHQ1 is added to the probe asa quencher that is used in combination with 6FAM to create a fluorogenichybridization probe for the real-time MS-PCR reaction. Amplificationswere done in 96-well plates in a CFX96 Real-Time System (Biorad) underthe following conditions: 95° C. for 10 minutes, followed by 50 cyclesof 30 sec at 95° C. and 60 sec at primer-specific annealing/extensiontemperature (see Table VII).

TABLE VII Primers used in this study. [+N] denotes Locked Nucleic Acid(LNA) bases. Primer/ Annealing/ Size Gene probe Primer/probe sequenceExtension (bp) VIM Forward TCGTTTCGAGGTTTTCGCGTTA 68° C. 217 PrimerGAGAC (SEQ ID NO: 62) Reverse CGACTAAAACTCGACCGACTCG PrimerCGA (SEQ ID NO: 24) Probe CGGGAGTTAGT[+T]CGCGTTATCGTCGTCGTTT (SEQ ID NO: 73) Actin Forward GGATAGGATAGTTTTATTTTTA 57° C.217 Primer G (SEQ ID NO: 74) Reverse ATACAAAACTATACTCAACCAA Primer(SEQ ID NO: 75) Probe 5′-ACCACCACCCAACACACAATAA CAAACACA-3′(SEQ ID NO: 76)

3. Vimentin Methylation in Esophageal Neoplasms and Precursor Lesions.

Evidence of vimentin methylation in esophageal adenocarcinomas (EAC) wasexamined. EAC primarily arises in the distal esophagus. Out of 18 casesof EAC examined, 15 cases (83%) showed high level vimentin methylation,of from 10%400% of total input tumor DNA (FIG. 51). As tumor tissuescontained both cancer and normal stromal elements, we interpret theselevels as suggesting vimentin methylation was likely present in all ofthe cancer cells. Two additional EAC cases showed lower levels ofvimentin methylation of between 1%-10%. No vimentin methylation wasdetected in this assay in any of 9 normal esophagus samples tested. Highlevel vimentin methylation was also detected in some squamous carcinomasof the esophagus, cancers that primarily arise in the upper esophagus,and for which 3 of 9 cases (33%) demonstrated vimentin methylation atlevels between 10%-100% of total tumor DNA.

To further interrogate the timing of vimentin methylation in esophagealneoplasias, we next examined samples of Barrett's Esophagus (BE), aprecursor lesion of neoplasias of the distal esophagus that canultimately give rise to EAC. As shown in FIG. 51, for 7 of 7 cases ofadvanced BE that showed high grade dysplasia, all had high levelvimentin methylation of from 10%-100%. High level vimentin methylationwas also detected in 12 of 13 (92%) cases of BE without dysplasia. Thesefindings suggest that vimentin methylation is an early and highly commonepigenetic change in the pathway of glandular neoplasia of theesophagus.

From 5 cases of EAC, additional concurrent biopsies were also availablefrom regions of esophagus that showed either simple BE or BE with highgrade dysplasia (FIG. 52). In each of these cases, the high level ofvimentin methylation detected in the EAC was also detected in theconcurrent BE and/or BE with high grade dysplasia tissue. This isconsistent with a model of vimentin methylation being an early eventpresent throughout the neoplastic clone, and of EAC arising from aninitiated field of BE tissue.

Current clinical practice provides for longitudinal endoscopicsurveillance of individuals with Barrett's esophagus. In 5 individualswith esophageal neoplasias that showed vimentin methylation, we wereable to identify additional tissue samples from prior biopsies ofBarrett's epithelium obtained from 1 to 7 years earlier in time. In 4 ofthese 5 cases, vimentin methylation was also detected as present in theearlier sample. (FIG. 53) In one individual, vimentin methylation wasdetected in each of 2 concurrent biopsies of BE, but was absent in thesame individual's BE sampled 4 years previously (Patient 10: FIG. 53 anddata not shown). These data further support a model which vimentinmethylation principally occurs as an early epigenetic event in thepathway of esophageal carcinogenesis, and in which EACs arise from aninitiated field of BE.

FIG. 55 summarizes additional results obtained by measuring DNAmethylation of vimentin in esophageal brushings of Barrett's Esophagus(BE), Barrett's Esophagus with High Grade Dysplasia (HGD), andEsophageal Adenocarcinoma (EAC).

4. Vimentin Methylation in Gastric Cancer

Similar to our findings in esophageal neoplasias, vimentin methylationalso proved commonly present in gastric cancers. High levels of vimentinmethylation (10%-100%) were identified in nine of eleven (82%) signetring gastric cancers and in four of 10 (40%) intestinal type gastriccancers (FIG. 54). The difference between these two gastric cancer typeswas not statistically significant (P=0.08). Low level vimentinmethylation was further detected in an additional 3 intestinal typecancers. Vimentin methylation was not detected in any of 5 normalgastric mucosa samples from cancer free individuals, and was also notdetected in 5 of 7 gastric mucosa samples from individuals whosestomachs did harbor gastric cancer. In the remaining 2 persons withgastric cancers, trace (<1%) vimentin methylation was detected in theaccompanying normal gastric mucosa. In these two cases this suggeststhat either the trace cancer cells were present in the normal mucosa, oralternatively, that the cancers may have developed from a field ofinitiated cells already marked by acquisition of vimentin methylation.Supporting this latter possibility was the finding that in oneindividual who harbored both a gastric cancer and a gastric dysplasia,both lesions were positive for vimentin methylation (data not shown).

In summary, the data presented above demonstrate that acquisition ofaberrant vimentin methylation is highly common in each of theseneoplasms or pre-neoplastic conditions (e.g., Barrett's esophagus,esophageal adenocarcinomas, and gastric cancers), and is largely absentfrom the corresponding normal tissues. Vimentin methylation was detectedin 92% of Barrett's esophagus, 100% of Barrett's esophagus with highgrade dysplasia, and 83% of adenocarcinomas of the esophagus. Vimentinmethylation similarly was detected in 82% of signet ring and 40% ofintestinal type gastric cancers. These findings establish aberrantvimentin methylation as a highly common epigenetic alteration inneoplasias that arise throughout the gut and also demonstrate thatBarrett's esophagus, even without dysplasia, already contains epigeneticalterations typical of adenocarcinomas of the esophagus. These findingsfurther demonstrate that vimentin methylation is a useful biomarker ofgastrointestinal neoplasia in both the upper and lower gastrointestinaltract.

4. Vimentin Methylation in Pancreatic Cancer

To determine the presence of aberrant vimentin methylation in pancreaticcancer, ten pancreatic cancer samples were obtained from formalin fixedparaffin embedded (FFPE) tissue samples. FFPE pancreatic cancer tissueswere sampled either as core samples from the FFPE tissue block (7/10) oras slides cut from the FFPE tissue block (3/10). Aberrant vimentinmethylation was detected in 3 of the core samples, and in all of theslides. The data is summarized in FIG. 56.

Incorporation by Reference

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

Equivalents

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1-36. (canceled)
 37. A method for determining a vimentin level in ahuman subject, comprising: a) obtaining a sample from a human subject;and b) assaying a vimentin nucleic acid in the sample for the presenceor absence of methylation within a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 2 and fragments thereof, and SEQ IDNOS:40-45.
 38. The method of claim 37, wherein the sample is a bodilyfluid selected from the group consisting of blood, serum, plasma, ablood-derived fraction, stool, urine, and a colonic effluent.
 39. Themethod of claim 38, wherein the bodily fluid is obtained from a subjectsuspected of having or is known to have colon neoplasia.
 40. The methodof claim 39, wherein said colon neoplasia is colon cancer.
 41. Themethod of claim 37, wherein the assay comprises methylation-specificPCR.
 42. The method of claim 41, comprising: a) treating DNA from thesample with a compound that converts non-methylated cytosine bases inthe DNA to a different base; b) amplifying a region of thecompound-converted vimentin nucleotide sequence with a forward primerand a reverse primer; and c) analyzing the methylation patterns of saidvimentin nucleotide sequences.
 43. The method of claim 41, comprising:a) treating DNA from the sample with a compound that convertsnon-methylated cytosine bases in the DNA to a different base; b)amplifying a region of the compound-converted vimentin nucleotidesequence with a forward primer and a reverse primer; and c) detectingthe presence and/or amount of the amplified product.
 44. The method ofclaim 41, wherein the compound used to treat DNA is a bisulfitecompound.
 45. The method of any of claim 37, wherein the assay comprisesusing a methylation-specific restriction enzyme.
 46. The method of claim45, wherein said methylation-specific restriction enzyme is selectedfrom HpaII, SmaI, SacII, EagI, MspI, BstUI, and BssHII.
 47. A method formonitoring vimentin levels in a human subject, comprising: a) assaying avimentin nucleic acid in a sample from the human subject for thepresence or absence of methylation within a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 2 and fragments thereof, andSEQ ID NOS:40-45 for a first time; and b) at a later time, assaying avimentin nucleic acid in another sample from the same human subject forthe presence or absence of methylation within a nucleotide sequenceassayed in step a).
 48. The method of claim 47, wherein each sample is abodily fluid selected from the group consisting of blood, serum, plasma,a blood-derived fraction, stool, urine, and a colonic effluent.
 49. Themethod of claim 48, wherein the bodily fluid is obtained from a subjectsuspected of having or is known to have colon neoplasia.
 50. The methodof claim 49, wherein said colon neoplasia is colon cancer.
 51. Themethod of claim 47, wherein the assay comprises methylation-specificPCR.
 52. The method of claim 51, comprising: a) treating DNA from asample with a compound that converts non-methylated cytosine bases inthe DNA to a different base; b) amplifying a region of thecompound-converted vimentin nucleotide sequence with a forward primerand a reverse primer; and c) analyzing the methylation patterns of saidvimentin nucleotide sequences.
 53. The method of claim 51, comprising:a) treating DNA from the sample with a compound that convertsnon-methylated cytosine bases in the DNA to a different base; b)amplifying a region of the compound-converted vimentin nucleotidesequence with a forward primer and a reverse primer; and c) detectingthe presence and/or amount of the amplified product.
 54. The method ofclaim 51, wherein the compound used to treat DNA is a bisulfitecompound.
 55. The method of claim 47, wherein the assay comprises usinga methylation-specific restriction enzyme.
 56. The method of claim 55,wherein said methylation-specific restriction enzyme is selected fromHpaII, SmaI, SacII, EagI, MspI, BstUI, and BssHII.